Chlamydia antigens and corresponding DNA fragments and uses thereof

ABSTRACT

The present invention provides a method of nucleic acid, including DNA, immunization of a host, including humans, against disease caused by infection by a strain of Chlamydia, specifically  C. pneumoniae,  employing a vector containing a nucleotide sequence encoding full-length, 5′-truncated or 3′-truncated 76 kDa protein of a strain of  Chlamydia pneumoniae  and a promoter to effect expression of the 76 kDa protein gene in the host. Modifications are possible within the scope of this invention.

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisionalapplication Ser. No. 60/132,270, filed May 3, 1999, and U.S. provisionalapplication Ser. No. 60/141,276 filed Jun. 30, 1999.

FIELD OF INVENTION

[0002] The present invention relates to the Chlamydia 76kDa protein andcorresponding DNA molecules, which can be used to prevent and treatChlamydia infection in mammals, such as humans.

BACKGROUND OF THE INVENTION

[0003] Chlamydiae are prokaryotes. They exhibit morphologic andstructural similarities to gram-negative bacteria including a trilaminarouter membrane, which contains lipopolysaccharide and several membraneproteins that are structurally and functionally analogous to proteinsfound in E coli. They are obligate intra-cellular parasites with aunique biphasic life cycle consisting of a metabolically inactive butinfectious extracellular stage and a replicating but non-infectiousintracellular stage. The replicative stage of the life-cycle takes placewithin a membrane-bound inclusion which sequesters the bacteria awayfrom the cytoplasm of the infected host cell.

[0004]C. pneumoniae is a common human pathogen, originally described asthe TWAR strain of Chlamydia psittaci but subsequently recognised to bea new species. C. pneumoniae is antigenically, genetically andmorphologically distinct from other Chlamydia species (C. trachomatis,C. pecorum and C. psittaci). It shows 10% or less DNA sequence homologywith either of C.trachomatis or C.psittaci.

[0005]C. pneumoniae is a common cause of community acquired pneumonia,only less frequent than Streptococcus pneumoniae and Mycoplasmapneumoniae (Grayston et al. (1995) Journal of Infectious Diseases168:1231; Campos et al. (1995) Investigation of Ophthalmology and VisualScience 36:1477). It can also cause upper respiratory tract symptoms anddisease, including bronchitis and sinusitis (Grayston et al. (1995)Journal of Infectious Diseases 168:1231; Grayston et al (1990) Journalof Infectious Diseases 161:618; Marrie (1993) Clinical InfectiousDiseases. 18:501; Wang et al (1986) Chlamydial infections CambridgeUniversity Press, Cambridge. p. 329. The great majority of the adultpopulation (over 60%) has antibodies to C. pneumoniae (Wang et al (1986)Chlamydial infections. Cambridge University Press, Cambridge. p. 329),indicating past infection which was unrecognized or asymptomatic.

[0006]C. pneumoniae infection usually presents as an acute respiratorydisease (i.e., cough, sore throat, hoarseness, and fever; abnormal chestsounds on auscultation). For most patients, the cough persists for 2 to6 weeks, and recovery is slow. In approximately 10% of these cases,upper respiratory tract infection is followed by bronchitis orpneumonia. Furthermore, during a C. pneumoniae epidemic, subsequentco-infection with pneumococcus has been noted in about half of thesepneumonia patients, particularly in the infirm and the elderly. As notedabove, there is more and more evidence that C. pneumoniae infection isalso linked to diseases other than respiratory infections.

[0007] The reservoir for the organism is presumably people. In contrastto C. psittaci infections, there is no known bird or animal reservoir.Transmission has not been clearly defined. It may result from directcontact with secretions, from fomites, or from airborne spread. There isa long incubation period, which may last for many months. Based onanalysis of epidemics, C. pneumoniae appears to spread slowly through apopulation (case-to-case interval averaging 30 days) because infectedpersons are inefficient transmitters of the organism. Susceptibility toC. pneumoniae is universal. Reinfections occur during adulthood,following the primary infection as a child. C. pneumoniae appears to bean endemic disease throughout the world, noteworthy for superimposedintervals of increased incidence (epidemics) that persist for 2 to 3years. C. trachomatis infection does not confer cross-immunity to C.pneumoniae. Infections are easily treated with oral antibiotics,tetracycline or erythromycin (2 g/d, for at least 10 to 14 d). Arecently developed drug, azithromycin, is highly effective as asingle-dose therapy against Chlamydial infections.

[0008] In most instances, C. pneumoniae infection is often mild andwithout complications, and up to 90% of infections are subacute orunrecognized. Among children in industrialized countries, infectionshave been thought to be rare up to the age of 5 y, although a recentstudy (E Normann et al, Chlamydia pneumoniae in children with acuterespiratory tract infections, Acta Paediatrica, 1998, Vol 87, Iss 1, pp23-27) has reported that many children in this age group show PCRevidence of infection despite being seronegative, and estimates aprevalence of 17-19% in 2-4 y olds. In developing countries, theseroprevalence of C. pneumoniae antibodies among young children iselevated, and there are suspicions that C. pneumoniae may be animportant cause of acute lower respiratory tract disease and mortalityfor infants and children in tropical regions of the world.

[0009] From seroprevalence studies and studies of local epidemics, theinitial C. pneumoniae infection usually happens between the ages of 5and 20 y. In the USA, for example, there are estimated to be 30,000cases of childhood pneumonia each year caused by C. pneumoniae.Infections may cluster among groups of children or young adults (e.g.,school pupils or military conscripts).

[0010]C. pneumoniae causes 10 to 25% of community-acquired lowerrespiratory tract infections (as reported from Sweden, Italy, Finland,and the USA). During an epidemic, C. pneumonia infection may account for50 to 60% of the cases of pneumonia. During these periods, also, moreepisodes of mixed infections with S. pneumoniae have been reported.

[0011] Reinfection during adulthood is common; the clinical presentationtends to be milder. Based on population seroprevalence studies, theretends to be increased exposure with age, which is particularly evidentamong men. Some investigators have speculated that a persistent,asymptomatic C. pneumoniae infection state is common.

[0012] In adults of middle age or older, C. pneumoniae infection mayprogress to chronic bronchitis and sinusitis. A study in the USArevealed that the incidence of pneumonia caused by C. pneumoniae inpersons younger than 60 years is 1 case per 1,000 persons per year; butin the elderly, the disease incidence rose three-fold. C. pneumoniaeinfection rarely leads to hospitalization, except in patients with anunderlying illness.

[0013] Of considerable importance is the association of atherosclerosisand C. pneumoniae infection. There are several epidemiological studiesshowing a correlation of previous infections with C. pneumoniae andheart attacks, coronary artery and carotid artery disease (Saikku et al.(1988) Lancet;ii:983; Thom et al. (1992) JAMA 268:68; Linnanmaki et al.(1993), Circulation 87:1030; Saikku et al. (1992)Annals InternalMedicine 116:273; Melnick et al(1993) American Journal of Medicine95:499). Moreover, the organisms has been detected in atheromas andfatty streaks of the coronary, carotid, peripheral arteries and aorta(Shor et al. (1992) South African. Medical Journal 82:158; Kuo et al.(1993) Journal of Infectious Diseases 167:841; Kuo et al. (1993)Arteriosclerosis and Thrombosis 13:1500; Campbell et al (1995) Journalof Infectious Diseases 172:585; Chiu et al. Circulation, 1997 (InPress)). Viable C. pneumoniae has been recovered from the coronary andcarotid artery (Ramirez et al (1996) Annals of Internal Medicine125:979; Jackson et al. Abst. K121, p272, 36^(th) ICAAC, 15-18 Sept.1996, New Orleans). Furthermore, it has been shown that C. pneumoniaecan induce changes of atherosclerosis in a rabbit model (Fong et al(1997) Journal of Clinical Microbiolology 35:48). Taken together, theseresults indicate that it is highly probable that C. pneumoniae can causeatherosclerosis in humans, though the epidemiological importance ofChlamydial atherosclerosis remains to be demonstrated.

[0014] A number of recent studies have also indicated an associationbetween C. pneumoniae infection and asthma. Infection has been linked towheezing, asthmatic bronchitis, adult-onset asthma and acuteexacerbations of asthma in adults, and small-scale studies have shownthat prolonged antibiotic treatment was effective at greatly reducingthe severity of the disease in some individuals (Hahn DL, et al.Evidence for Chlamydia pneumoniae infection in steroid-dependentasthma.Ann Allergy Asthma Immunol. 1998 Jan; 80(1): 45-49.; Hahn DL, etal. Association of Chlamydia pneumoniae IgA antibodies with recentlysymptomatic asthma. Epidemiol Infect. 1996 Dec; 117(3): 513-517;Bjornsson E, et al. Serology of Chlamydia in relation to asthma andbronchial hyperresponsiveness. Scand J Infect Dis. 1996; 28(1): 63-69.;Hahn DL. Treatment of Chlamydia pneumoniae infection in adult asthma: abefore-after trial. J Fam Pract. 1995 Oct.; 41(4): 345-351.; Allegra L,et al. Acute exacerbations of asthma in adults: role of Chlamydiapneumoniae infection. Eur Respir J. 1994 Dec.; 7(12): 2165-2168.; HahnDL, et al. Association of Chlamydia pneumoniae (strain TWAR) infectionwith wheezing, asthmatic bronchitis, and adult-onset asthma. JAMA. Jul.10, 1991; 266(2): 225-230).

[0015] In light of these results a protective vaccine against C.pneumoniae infection would be of considerable importance. There is notyet an effective vaccine for any human Chlamydial infection. It isconceivable that an effective vaccine can be developed using physicallyor chemically inactivated Chlamydiae. However, such a vaccine does nothave a high margin of safety. In general, safer vaccines are made bygenetically manipulating the organism by attenuation or by recombinantmeans. Accordingly, a major obstacle in creating an effective and safevaccine against human Chlamydial infection has been the paucity ofgenetic information regarding Chlamydia, specifically C. pneumoniae.

[0016] Studies with C. trachomatis and C. psittaci indicate that safeand effective vaccine against Chlamydia is an attainable goal. Forexample, mice which have recovered from a lung infection with C.trachomatis are protected from infertility induced by a subsequentvaginal challenge (Pal et al. (1996) Infection and Immunity.64:5341).Similarly, sheep immunized with inactivated C. psittaci were protectedfrom subsequent Chlamydial-induced abortions and stillbirths (Jones etal. (1995) Vaccine 13:715). Protection from Chlamydial infections hasbeen associated with Th1 immune responses, particularly the induction ofINFg—producing CD4+T-cells (Igietsemes et al. (1993) Immunology 5:317).The adoptive transfer of CD4+ cell lines or clones to nude or SCID miceconferred protection from challenge or cleared chronic disease(Igietseme et al (1993) Regional Immunology 5:317; Magee et al (1993)Regional Immunology 5: 305), and in vivo depletion of CD4+ T cellsexacerbated disease post-challenge (Landers et al (1991) Infection &Immunity 59:3774; Magee et al (1995) Infection & Immunity 63:516).However, the presence of sufficiently high titres of neutralisingantibody at mucosal surfaces can also exert a protective effect (Cotteret al. (1995) Infection and Immunity 63:4704).

[0017] Antigenic variation within the species C. pneumoniae is not welldocumented due to insufficient genetic information, though variation isexpected to exist based on C. trachomatis. Serovars of C. trachomatisare defined on the basis of antigenic variation in the major outermembrane protein (MOMP), but published C. pneumoniae MOMP gene sequencesshow no variation between several diverse isolates of the organism(Campbell et al (1990) Infection and Immunity 58:93; McCafferty et al(1995) Infection and Immunity 63:2387-9; Knudsen et al (1996) ThirdMeeting of the European Society for Chlamydia Research, Vienna). Melgosaet al. (Infect. Immun. 1994. 62:880) claimed to have cloned the geneencoding a 76 kDa antigen from a single strain of C. pneumoniae. Anoperon encoding the 9 kDa and 9 kDa cyteine-rich outer membrane proteingenes has been described (Watson et al., Nucleic Acids Res (1990)18:5299; Watson et al., Microbiology (1995) 141:2489). Many antigensrecognized by immune sera to C. pneumoniae are conserved across allChlamydiae, but 98 kDa, 76 kDa and several other proteins may be C.pneumoniae-specific (Perez Melgosa et al., Infect. Immun. 1994. 62:880;Melgosa et al., FEMS Microbiol Lett (1993) 112 :199;, Campbell et al., JClin Microbiol (1990) 28 :1261; Iijima et al., J Clin Microbiol (1994)32:583). An assessment of the number and relative frequency of any C.pneumoniae serotypes, and the defining antigens, is not yet possible.The entire genome sequence of C. pneumoniae strain CWL-029 is now known(http://chlamydia-www.berkeley.edu:4231/) and as further sequencesbecome available a better understanding of antigenic variation may begained.

[0018] Many antigens recognised by immune sera to C. pneumoniae areconserved across all Chlamydiae, but 98 kDa, 76 kDa and 54 kDa proteinsappear to be C. pneumoniae-specific (Campos et al. (1995) Investigationof Ophthalmology and Visual Science 36:1477; Marrie (1993) ClinicalInfectious Diseases. 18:501; Wiedmann-Al-Ahmad M, et al. Reactions ofpolyclonal and neutralizing anti-p54 monoclonal antibodies with anisolated, species-specific 54-kilodalton protein of Chlamydiapneumoniae. Clin Diagn Lab Immunol. 1997 Nov; 4(6): 700-704).

[0019] Immunoblotting of isolates with sera from patients does showvariation of blotting patterns between isolates, indicating thatserotypes C. pneumoniae may exist (Grayston et al. (1995) Journal ofInfectious Diseases 168:1231; Ramirez et al (1996) Annals of InternalMedicine 125:979). However, the results are potentially confounded bythe infection status of the patients, since immunoblot profiles of apatient's sera change with time post-infection. An assessment of thenumber and relative frequency of any serotypes, and the definingantigens, is not yet possible.

[0020] Accordingly, a need exists for identifying and isolatingpolynucleotide sequences of C. pneumoniae for use in preventing andtreating Chlamydia infection.

SUMMARY OF THE INVENTION

[0021] The present invention provides purified and isolatedpolynucleotide molecules that encode the Chlamydia polypeptidedesignated 76 kDa protein (SEQ ID No: 1) which can be used in methods toprevent, treat, and diagnose Chlamydia infection. In one form of theinvention, the polynucleotide molecules are DNA that encode thepolypeptide of SEQ ID No: 2.

[0022] Another form of the invention provides polypeptides correspondingto the isolated DNA molecules. The amino acid sequence of thecorresponding encoded polypeptide is shown as SEQ ID No: 2.

[0023] Another form of the invention provides truncated polypeptidescorresponding to truncated DNA molecules. In one embodiment, thetruncated nucleotide and amino acid sequences are shown as SEQ ID Nos: 3and 4 respectively. In another embodiment, the truncated nucleotide andamino acid sequences are shown as SEQ ID Nos: 5 and 6 respectively.

[0024] Although Melgosa et al. has reported cloning a 76 kDa proteinfrom C. pneumoniae, comparison of the gene sequence as reported byMelgosa et al. to the published geneome sequence of C. pneumoniae(http://chlamydia-www.berkeley.edu:4231/) reveals that, in fact, thegenomic sequence in this region contains at least two open readingframes (ORFs), one in the 5′ portion and one in the 3′ portion. Thesequence reported in Melgosa et al. is an in-frame fusion of the 5′ endof the 5′ ORF. Thus, Melgosa's deduced protein is merely a 76 kDa fusionprotein and not the 76 kDa protein observed by immunoblotting fromvarious C. pneumoniae isolates. By contrast, the 76 kDa protein of thepresent invention is the full-length protein encoded by the 3′ ORF inthis region of the genome. Notably, further analysis of the genomesequence (http://chlamydia-www.berkeley.edu:4231/) reveals at least onein-frame ATG upstream of the start codon of the 5′ ORF, suggesting thatthe 5′ ORF may form part of one or more larger ORFs.

[0025] Those skilled in the art will readily understand that theinvention, having provided the polynucleotide sequences encoding theChlamydia 76 kDa protein, also provides polynucleotides encodingfragments derived from such a polypeptide. Moreover, the invention isunderstood to provide mutants and derivatives of such polypeptides andfragments derived therefrom, which result from the addition, deletion,or substitution of non-essential amino acids as described herein. Thoseskilled in the art would also readily understand that the invention,having provided the polynucleotide sequences encoding Chlamydiapolypeptides, further provides monospecific antibodies that specificallybind to such polypeptides.

[0026] The present invention has wide application and includesexpression cassettes, vectors, and cells transformed or transfected withthe polynucleotides of the invention. Accordingly, the present inventionfurther provides (i) a method for producing a polypeptide of theinvention in a recombinant host system and related expression cassettes,vectors, and transformed or transfected cells; (ii) a vaccine, or a livevaccine vector such as a pox virus, Salmonella typhimurium, or Vibriocholerae vector, containing a polynucleotide of the invention, suchvaccines and vaccine vectors being useful for, e.g., preventing andtreating Chlamydia infection, in combination with a diluent or carrier,and related pharmaceutical compositions and associated therapeuticand/or prophylactic methods; (iii) a therapeutic and/or prophylactic useof an RNA or DNA molecule of the invention, either in a naked form orformulated with a delivery vehicle, a polypeptide or combination ofpolypeptides, or a monospecific antibody of the invention, and relatedpharmaceutical compositions; (iv) a method for diagnosing the presenceof Chlamydia in a biological sample, which can involve the use of a DNAor RNA molecule, a monospecific antibody, or a polypeptide of theinvention; and (v) a method for purifying a polypeptide of the inventionby antibody-based affinity chromatography.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further understood from thefollowing description with reference to embodiments shown in thedrawings, in which:

[0028]FIG. 1 shows the full-length nucleotide sequence of the 76 kDaprotein gene (SEQ ID No: 1) and the deduced amino acid sequence of the76 kDa protein from Chlamydia pneumoniae (SEQ ID No: 2).

[0029]FIG. 2 shows the restriction enzyme analysis of the C. pneumoniae76 kDa protein gene.

[0030]FIG. 3 shows the nucleotide sequence containing a 3′-truncated 76kDa protein gene and its corresponding deduced amino acid sequence fromChlamydia pneumoniae; (note that nucleotides 1 to 665 and 2122 to 2238are unrelated to the 76 kDa protein gene).

[0031]FIG. 4 shows the construction and elements of plasmid pCACPNM555a,containing the full-length 76 kDa gene.

[0032]FIG. 5 shows the construction and elements of plasmid pCAI555,containing a 5′-truncated version of the 76 kDa gene.

[0033]FIG. 6 shows the construction and elements of plasmid pCAD76kDa,containing a 3′-truncated version of the 76 kDa gene from FIG. 3.

[0034]FIG. 7 illustrates protection against C. pneumoniae infection bypCACPNM555a following DNA immunization.

[0035]FIG. 8 illustrates protection against C. pneumoniae infection bypCAI555 following DNA immunization.

[0036]FIG. 9 illustrates protection against C. pneumoniae infection bypCAD76kDa following DNA immunization. For FIGS. 7 to 9, individual datapoints are shown for each animal (hollow diamonds) as well as mean andstandard deviations for each group (solid squares).

DETAILED DESCRIPTION OF INVENTION

[0037] The invention is described with reference to the followingsequences which are embodiments of the invention: SEQ ID NO: 1 is thefull-length sequence of the 76 kDa protein gene.

[0038] SEQ ID NO: 2 is the deduced full-length amino acid sequence ofthe 76 kDa protein.

[0039] SEQ ID NO: 3 is the 5′-truncated nucleotide sequence of the 76kDa protein gene.

[0040] SEQ ID NO: 4 is the 5′-truncated amino acid sequence of the 76kDa protein.

[0041] SEQ ID NO: 5 is the 3′-truncated nucleotide sequence of the 76kDa protein gene.

[0042] SEQ ID NO: 6 is the 3′-truncated amino acid sequence of the 76kDa protein, which forms the basis for immunoprotection by pCAD76kDa inFIG. 9.

[0043] SEQ ID NO: 7 is the sequence encoding a polypeptide containing atruncated 76 kDa protein. Using this sequence as a template, a fragmentwas amplified by PCR to form part of construct pCAD76kDa.

[0044] SEQ ID NO: 8 is the deduced amino acid sequence of a truncated 76kDa protein, as expressed from pCAD76kDa.

[0045] SEQ ID NO: 9 is the 5′ primer used to clone the full-length 76kDa protein gene and to amplify the full-length 76 kDa protein gene forpCACPNM555a.

[0046] SEQ ID NO: 10 is the 3′ primer used to clone the full-length 76kDa protein gene and to amplify the full-length 76 kDa protein gene forpCACPNM555a.

[0047] SEQ ID NO: 11 is the 5′ primer used to amplify the 5′-truncated76 kDa protein gene fragment for pCAI555.

[0048] SEQ ID NO: 12 is the 3′ primer used to amplify the 5′-truncated76 kDa protein gene fragment for pCAI555.

[0049] SEQ ID NO: 13 is the 5′ primer used to amplify the 3′-truncated76 kDa protein gene fragment for pCAD76kDa.

[0050] SEQ ID NO: 14 is the 3′ primer used to amplify the truncated 76kDa protein gene fragment for pCAD76kDa.

[0051] An open reading frame (ORF) encoding the Chlamydial 76 kDaprotein has been identified from the C. pneumoniae genome. The geneencoding this protein and its fragments have been inserted intoexpression plasmids and shown to confer immune protection againstChlamydial infection. Accordingly, this 76 kDa protein and relatedpolypeptides can be used to prevent and treat Chlamydia infection.

[0052] According to a first aspect of the invention, isolatedpolynucleotides are provided which encode Chlamydia polypeptides, whoseamino acid sequences are shown in SEQ ID Nos: 2, 4 and 6.

[0053] The term “isolated polynucleotide” is defined as a polynucleotideremoved from the environment in which it naturally occurs. For example,a naturally-occurring DNA molecule present in the genome of a livingbacteria or as part of a gene bank is not isolated, but the samemolecule separated from the remaining part of the bacterial genome, as aresult of, e.g., a cloning event (amplification), is isolated.Typically, an isolated DNA molecule is free from DNA regions (e.g.,coding regions) with which it is immediately contiguous at the 5′ or 3′end, in the naturally occurring genome. Such isolated polynucleotidesmay be part of a vector or a composition and still be defined asisolated in that such a vector or composition is not part of the naturalenvironment of such polynucleotide.

[0054] The polynucleotide of the invention is either RNA or DNA (cDNA,genomic DNA, or synthetic DNA), or modifications, variants, homologs orfragments thereof. The DNA is either double-stranded or single-stranded,and, if single-stranded, is either the coding strand or the non-coding(anti-sense) strand. Any one of the sequences that encode thepolypeptides of the invention as shown in SEQ ID No: 1, 3 or 5 is (a) acoding sequence, (b) a ribonucleotide sequence derived fromtranscription of (a), or (c) a coding sequence which uses the redundancyor degeneracy of the genetic code to encode the same polypeptides. By“polypeptide” or “protein” is meant any chain of amino acids, regardlessof length or post-translational modification (e.g., glycosylation orphosphorylation). Both terms are used interchangeably in the presentapplication.

[0055] Consistent with the first aspect of the invention, amino acidsequences are provided which are homologous to SEQ ID No: 2, 4 or 6. Asused herein, “homologous amino acid sequence” is any polypeptide whichis encoded, in whole or in part, by a nucleic acid sequence whichhybridizes at 25-35° C. below critical melting temperature (Tm), to anyportion of the nucleic acid sequence of SEQ ID No: 1, 3 or 5. Ahomologous amino acid sequence is one that differs from an amino acidsequence shown in SEQ ID No: 2, 4 or 6 by one or more conservative aminoacid substitutions. Such a sequence also encompass serotypic variants(defined below) as well as sequences containing deletions or insertionswhich retain inherent characteristics of the polypeptide such asimmunogenicity. Preferably, such a sequence is at least 75%, morepreferably 80%, and most preferably 90% identical to SEQ ID No: 2, 4 or6.

[0056] Homologous amino acid sequences include sequences that areidentical or substantially identical to SEQ ID No: 2, 4 or 6. By “aminoacid sequence substantially identical” is meant a sequence that is atleast 90%, preferably 95%, more preferably 97%, and most preferably 99%identical to an amino acid sequence of reference and that preferablydiffers from the sequence of reference by a majority of conservativeamino acid substitutions.

[0057] Conservative amino acid substitutions are substitutions amongamino acids of the same class. These classes include, for example, aminoacids having uncharged polar side chains, such as asparagine, glutamine,serine, threonine, and tyrosine; amino acids having basic side chains,such as lysine, arginine, and histidine; amino acids having acidic sidechains, such as aspartic acid and glutamic acid; and amino acids havingnonpolar side chains, such as glycine, alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan, andcysteine.

[0058] Homology is measured using sequence analysis software such asSequence Analysis Software Package of the Genetics Computer Group,University of Wisconsin Biotechnology Center, 1710 University Avenue,Madison, Wis. 53705. Amino acid sequences are aligned to maximizeidentity. Gaps may be artificially introduced into the sequence toattain proper alignment. Once the optimal alignment has been set up, thedegree of homology is established by recording all of the positions inwhich the amino acids of both sequences are identical, relative to thetotal number of positions.

[0059] Homologous polynucleotide sequences are defined in a similar way.Preferably, a homologous sequence is one that is at least 45%, morepreferably 60%, and most preferably 85% identical to the coding sequenceof SEQ ID No: 1, 3 or 5.

[0060] Consistent with the first aspect of the invention, polypeptideshaving a sequence homologous to SEQ ID No: 2, 4 or 6 includenaturally-occurring allelic variants, as well as mutants or any othernon-naturally occurring variants that retain the inherentcharacteristics of the polypeptide of SEQ ID No: 2, 4 or 6.

[0061] As is known in the art, an allelic variant is an alternate formof a polypeptide that is characterized as having a substitution,deletion, or addition of one or more amino acids that does not alter thebiological function of the polypeptide. By “biological function” ismeant the function of the polypeptide in the cells in which it naturallyoccurs, even if the function is not necessary for the growth or survivalof the cells. For example, the biological function of a porin is toallow the entry into cells of compounds present in the extracellularmedium. Biological function is distinct from antigenic property. Apolypeptide can have more than one biological function.

[0062] Allelic variants are very common in nature. For example, abacterial species such as C. pneumoniae, is usually represented by avariety of strains that differ from each other by minor allelicvariations. Indeed, a polypeptide that fulfills the same biologicalfunction in different strains can have an amino acid sequence (andpolynucleotide sequence) that is not identical in each of the strains.Despite this variation, an immune response directed generally againstmany allelic variants has been demonstrated. In studies of theChlamydial MOMP antigen, cross-strain antibody binding plusneutralization of infectivity occurs despite amino acid sequencevariation of MOMP from strain to strain, indicating that the MOMP, whenused as an immunogen, is tolerant of amino acid variations.

[0063] Polynucleotides encoding homologous polypeptides or allelicvariants are retrieved by polymerase chain reaction (PCR) amplificationof genomic bacterial DNA extracted by conventional methods. Thisinvolves the use of synthetic oligonucleotide primers matching upstreamand downstream of the 5′ and 3′ ends of the encoding domain. Suitableprimers are designed according to the nucleotide sequence informationprovided in SEQ ID No:1, 3 or 5. The procedure is as follows: a primeris selected which consists of 10 to 40, preferably 15 to 25 nucleotides.It is advantageous to select primers containing C and G nucleotides in aproportion sufficient to ensure efficient hybridization; i.e., an amountof C and G nucleotides of at least 40%, preferably 50% of the totalnucleotide content. A standard PCR reaction contains typically 0.5 to 5Units of Taq DNA polymerase per 100 μL, 20 to 200 μM deoxynucleotideeach, preferably at equivalent concentrations, 0.5 to 2.5 mM magnesiumover the total deoxynucleotide concentration, 10⁵ to 10⁶ targetmolecules, and about 20 pmol of each primer. About 25 to 50 PCR cyclesare performed, with an annealing temperature 15° C. to 5° C. below thetrue Tm of the primers. A more stringent annealing temperature improvesdiscrimination against incorrectly annealed primers and reducesincorporation of incorrect nucleotides at the 3′ end of primers. Adenaturation temperature of 95° C. to 97° C. is typical, although highertemperatures may be appropriate for dematuration of G+C-rich targets.The number of cycles performed depends on the starting concentration oftarget molecules, though typically more than 40 cycles is notrecommended as non-specific background products tend to accumulate.

[0064] An alternative method for retrieving polynucleotides encodinghomologous polypeptides or allelic variants is by hybridizationscreening of a DNA or RNA library. Hybridization procedures arewell-known in the art and are described in Ausubel et al., (Ausubel etal., Current Protocols in Molecular Biology, John Wiley & Sons Inc.,1994), Silhavy et al. (Silhavy et al. Experiments with Gene Fusions,Cold Spring Harbor Laboratory Press, 1984), and Davis et al. (Davis etal. A Manual for Genetic Engineering: Advanced Bacterial Genetics, ColdSpring Harbor Laboratory Press, 1980)). Important parameters foroptimizing hybridization conditions are reflected in a formula used toobtain the critical melting temperature above which two complementaryDNA strands separate from each other (Casey & Davidson, Nucl. Acid Res.(1977) 4:1539). For polynucleotides of about 600 nucleotides or larger,this formula is as follows: Tm=81.5+0.41×(% G+C) +16.6 log (cation ionconcentration)−0.63×(% formamide)−600/base number. Under appropriatestringency conditions, hybridization temperature (Th) is approximately20 to 40° C., 20 to 25° C., or, preferably 30 to 40° C. below thecalculated Tm. Those skilled in the art will understand that optimaltemperature and salt conditions can be readily determined.

[0065] For the polynucleotides of the invention, stringent conditionsare achieved for both pre-hybridizing and hybridizing incubations (i)within 4-16 hours at 42° C., in 6×SSC containing 50% formamide, or (ii)within 4-16 hours at 65° C. in an aqueous 6×SSC solution (1 M NaCl, 0.1M sodium citrate (pH 7.0)). Typically, hybridization experiments areperformed at a temperature from 60 to 68° C., e.g. 65° C. At such atemperature, stringent hybridization conditions can be achieved in6×SSC, preferably in 2×SSC or 1×SSC, more preferably in 0.5×SSc, 0.3×SSCor 0.1×SSC (in the absence of formamide). 1×SSC contains 0.15 M NaCl and0.015 M sodium citrate.

[0066] Useful homologs and fragments thereof that do not occur naturallyare designed using known methods for identifying regions of an antigenthat are likely to tolerate amino acid sequence changes and/ordeletions. As an example, homologous polypeptides from different speciesare compared; conserved sequences are identified. The more divergentsequences are the most likely to tolerate sequence changes. Homologyamong sequences may be analyzed using, as an example, the BLAST homologysearching algorithm of Altschul et al., Nucleic Acids Res.; 25:3389-3402(1997). Alternatively, sequences are modified such that they become morereactive to T- and/or B-cells, based on computer-assisted analysis ofprobable T- or B-cell epitopes Yet another alternative is to mutate aparticular amino acid residue or sequence within the polypeptide invitro, then screen the mutant polypeptides for their ability to preventor treat Chlamydia infection according to the method outlined below.

[0067] A person skilled in the art will readily understand that byfollowing the screening process of this invention, it will be determinedwithout undue experimentation whether a particular homolog of SEQ ID No:2, 4 or 6 may be useful in the prevention or treatment of Chlamydiainfection. The screening procedure comprises the steps:

[0068] (i) immunizing an animal, preferably mouse, with the test homologor fragment;

[0069] (ii) inoculating the immunized animal with Chlamydia;

[0070] and

[0071] (iii) selecting those homologs or fragments which conferprotection against Chlamydia.

[0072] By “conferring protection” is meant that there is a reduction inseverity of any of the effects of Chlamydia infection, in comparisonwith a control animal which was not immunized with the test homolog orfragment.

[0073] Consistent with the first aspect of the invention, polypeptidederivatives are provided that are partial sequences of SEQ ID No: 2, 4or 6, partial sequences of polypeptide sequences homologous to SEQ IDNo: 2, 4 or 6, polypeptides derived from full-length polypeptides byinternal deletion, and fusion proteins.

[0074] It is an accepted practice in the field of immunology to usefragments and variants of protein immunogens as vaccines, as all that isrequired to induce an immune response to a protein is a small (e.g., 8to 10 amino acid) immunogenic region of the protein. Various shortsynthetic peptides corresponding to surface-exposed antigens ofpathogens other than Chlamydia have been shown to be effective vaccineantigens against their respective pathogens, e.g. an 11 residue peptideof murine mammary tumor virus (Casey & Davidson, Nucl. Acid Res. (1977)4:1539), a 16-residue peptide of Semliki Forest virus (Snijders et al.,1991. J. Gen. Virol. 72:557-565), and two overlapping peptides of 15residues each from canine parvovirus (Langeveld et al., Vaccine12(15):1473-1480, 1994).

[0075] Accordingly, it will be readily apparent to one skilled in theart, having read the present description, that partial sequences of SEQID No: 2, 4 or 6 or their homologous amino acid sequences are inherentto the full-length sequences and are taught by the present invention.Such polypeptide fragments preferably are at least 12 amino acids inlength. Advantageously, they are at least 20 amino acids, preferably atleast 50 amino acids, and more preferably at least 75 amino acids andmost preferably at least 100 amino acids in length.

[0076] Polynucleotides of 30 to 600 nucleotides encoding partialsequences of sequences homologous to SEQ ID No: 2, 4 or 6 are retrievedby PCR amplification using the parameters outlined above and usingprimers matching the sequences upstream and downstream of the 5′ and 3′ends of the fragment to be amplified. The template polynucleotide forsuch amplification is either the full length polynucleotide homologousto SEQ ID No: 1, 3 or 5, or a polynucleotide contained in a mixture ofpolynucleotides such as a DNA or RNA library. As an alternative methodfor retrieving the partial sequences, screening hybridization is carriedout under conditions described above and using the formula forcalculating Tm. Where fragments of 30 to 600 nucleotides are to beretrieved, the calculated Tm is corrected by subtracting(600/polynucleotide size in base pairs) and the stringency conditionsare defined by a hybridization temperature that is 5 to 10° C. below Tm.Where oligonucleotides shorter than 20-30 bases are to be obtained, theformula for calculating the Tm is as follows: Tm=4×(G+C)+2 (A+T). Forexample, an 18 nucleotide fragment of 50% G+C would have an approximateTm of 54° C. Short peptides that are fragments of SEQ ID No: 2, 4 or 6or its homologous sequences, are obtained directly by chemical synthesis(E. Gross and H. J. Meinhofer, 4 The Peptides: Analysis, Synthesis,Biology; Modern Techniques of Peptide Synthesis, John Wiley & Sons(1981), and M. Bodanzki, Principles of Peptide Synthesis,Springer-Verlag (1984)).

[0077] Useful polypeptide derivatives, e.g., polypeptide fragments, aredesigned using computer-assisted analysis of amino acid sequences. Thiswould identify probable surface-exposed, antigenic regions (Hughes etal., 1992. Infect. Immun. 60(9):3497). Analysis of 6 amino acidsequences contained in SEQ ID No: 2, 4 or 6, based on the product offlexibility and hydrophobicity propensities using the program SEQSEE(Wishart DS, et al. “SEQSEE: a comprehensive program suite for proteinsequence analysis.” Comput Appl Biosci. 1994 Apr;10(2):121-32), canreveal potential B- and T-cell epitopes which may be used as a basis forselecting useful immunogenic fragments and variants. This analysis usesa reasonable combination of external surface features that is likely tobe recognized by antibodies. Probable T-cell epitopes for HLA-A0201 MHCsubclass may be revealed by an algorithms that emulate an approachdeveloped at the NIH (Parker KC, et al. “Peptide binding to MHC class Imolecules: implications for antigenic peptide prediction.” Immunol Res1995;14(l):34-57).

[0078] Epitopes which induce a protective T cell-dependent immuneresponse are present throughout the length of the polypeptide. However,some epitopes may be masked by secondary and tertiary structures of thepolypeptide. To reveal such masked epitopes large internal deletions arecreated which remove much of the original protein structure and exposesthe masked epitopes. Such internal deletions sometimes effect theadditional advantage of removing immunodominant regions of highvariability among strains.

[0079] Polynucleotides encoding polypeptide fragments and polypeptideshaving large internal deletions are constructed using standard methods(Ausubel et al., Current Protocols in Molecular Biology, John Wiley &Sons Inc., 1994). Such methods include standard PCR, inverse PCR,restriction enzyme treatment of cloned DNA molecules, or the method ofKunkel et al. (Kunkel et al. Proc. Natl. Acad. Sci. USA (1985) 82:448).Components for these methods and instructions for their use are readilyavailable from various commercial sources such as Stratagene. Once thedeletion mutants have been constructed, they are tested for theirability to prevent or treat Chlamydia infection as described above.

[0080] As used herein, a fusion polypeptide is one that contains apolypeptide or a polypeptide derivative of the invention fused at the N-or C-terminal end to any other polypeptide (hereinafter referred to as apeptide tail). A simple way to obtain such a fusion polypeptide is bytranslation of an in-frame fusion of the polynucleotide sequences, i.e.,a hybrid gene. The hybrid gene encoding the fusion polypeptide isinserted into an expression vector which is used to transform ortransfect a host cell. Alternatively, the polynucleotide sequenceencoding the polypeptide or polypeptide derivative is inserted into anexpression vector in which the polynucleotide encoding the peptide tailis already present. Such vectors and instructions for their use arecommercially available, e.g. the pMal-c2 or pMal-p2 system from NewEngland Biolabs, in which the peptide tail is a maltose binding protein,the glutathione-S-transferase system of Pharmacia, or the His-Tag systemavailable from Novagen. These and other expression systems provideconvenient means for further purification of polypeptides andderivatives of the invention.

[0081] An advantageous example of a fusion polypeptide is one where thepolypeptide or homolog or fragment of the invention is fused to apolypeptide having adjuvant activity, such as subunit B of eithercholera toxin or E. coli heat-labile toxin. Another advantageous fusionis one where the polypeptide, homolog or fragment is fused to a strongT-cell epitope or B-cell epitope. Such an epitope may be one known inthe art (e.g. the Hepatitis B virus core antigen, D. R. Millich et al.,“Antibody production to the nucleocapsid and envelope of the Hepatitis Bvirus primed by a single synthetic T cell site”, Nature. 1987.329:547-549), or one which has been identified in another polypeptide ofthe invention based on computer-assisted analysis of probable T- orB-cell epitopes. Consistent with this aspect of the invention is afusion polypeptide comprising T- or B-cell epitopes from SEQ ID No: 2, 4or 6 or its homolog or fragment, wherein the epitopes are derived frommultiple variants of said polypeptide or homolog or fragment, eachvariant differing from another in the location and sequence of itsepitope within the polypeptide. Such a fusion is effective in theprevention and treatment of Chlamydia infection since it optimizes theT- and B-cell response to the overall polypeptide, homolog or fragment.

[0082] To effect fusion, the polypeptide of the invention is fused tothe N-, or preferably, to the C-terminal end of the polypeptide havingadjuvant activity or T- or B-cell epitope. Alternatively, a polypeptidefragment of the invention is inserted internally within the amino acidsequence of the polypeptide having adjuvant activity. The T- or B-cellepitope may also be inserted internally within the amino acid sequenceof the polypeptide of the invention.

[0083] Consistent with the first aspect, the polynucleotides of theinvention also encode hybrid precursor polypeptides containingheterologous signal peptides, which mature into polypeptides of theinvention. By “heterologous signal peptide” is meant a signal peptidethat is not found in naturally-occurring precursors of polypeptides ofthe invention.

[0084] Polynucleotide molecules according to the invention, includingRNA, DNA, or modifications or combinations thereof, have variousapplications. A DNA molecule is used, for example, (i) in a process forproducing the encoded polypeptide in a recombinant host system, (ii) inthe construction of vaccine vectors such as poxviruses, which arefurther used in methods and compositions for preventing and/or treatingChlamydia infection, (iii) as a vaccine agent (as well as an RNAmolecule), in a naked form or formulated with a delivery vehicle and,(iv) in the construction of attenuated Chlamydia strains that canover-express a polynucleotide of the invention or express it in anon-toxic, mutated form.

[0085] Accordingly, a second aspect of the invention encompasses (i) anexpression cassette containing a DNA molecule of the invention placedunder the control of the elements required for expression, in particularunder the control of an appropriate promoter; (ii) an expression vectorcontaining an expression cassette of the invention; (iii) a procaryoticor eucaryotic cell transformed or transfected with an expressioncassette and/or vector of the invention, as well as (iv) a process forproducing a polypeptide or polypeptide derivative encoded by apolynucleotide of the invention, which involves culturing a procaryoticor eucaryotic cell transformed or transfected with an expressioncassette and/or vector of the invention, under conditions that allowexpression of the DNA molecule of the invention and, recovering theencoded polypeptide or polypeptide derivative from the cell culture.

[0086] A recombinant expression system is selected from procaryotic andeucaryotic hosts. Eucaryotic hosts include yeast cells (e.g.,Saccharomyces cerevisiae or Pichia pastoris), mammalian cells (e.g.,COS1, NIH3T3, or JEG3 cells), arthropods cells (e.g., Spodopterafrugiperda (SF9) cells), and plant cells. A preferred expression systemis a procaryotic host such as E. coli. Bacterial and eucaryotic cellsare available from a number of different sources including commercialsources to those skilled in the art, e.g., the American Type CultureCollection (ATCC; Rockville, Md.). Commercial sources of cells used forrecombinant protein expression also provide instructions for usage ofthe cells.

[0087] The choice of the expression system depends on the featuresdesired for the expressed polypeptide. For example, it may be useful toproduce a polypeptide of the invention in a particular lipidated form orany other form.

[0088] One skilled in the art would redily understand that not allvectors and expression control sequences and hosts would be expected toexpress equally well the polynucleotides of this invention. With theguidelines described below, however, a selection of vectors, expressioncontrol sequences and hosts may be made without undue experimentationand without departing from the scope of this invention.

[0089] In selecting a vector, the host must be chosen that is compatiblewith the vector which is to exist and possibly replicate in it.Considerations are made with respect to the vector copy number, theability to control the copy number, expression of other proteins such asantibiotic resistance. In selecting an expression control sequence, anumber of variables are considered. Among the important variable are therelative strength of the sequence (e.g. the ability to drive expressionunder various conditions), the ability to control the sequence'sfunction, compatibility between the polynucleotide to be expressed andthe control sequence (e.g. secondary structures are considered to avoidhairpin structures which prevent efficient transcription). In selectingthe host, unicellular hosts are selected which are compatible with theselected vector, tolerant of any possible toxic effects of the expressedproduct, able to secrete the expressed product efficiently if such isdesired, to be able to express the product in the desired conformation,to be easily scaled up, and to which ease of purification of the finalproduct.

[0090] The choice of the expression cassette depends on the ost systemselected as well as the features desired for the xpressed polypeptide.Typically, an expression cassette includes a promoter that is functionalin the selected host system and can be constitutive or inducible; aribosome binding site; a start codon (ATG) if necessary; a regionencoding a signal peptide, e.g., a lipidation signal peptide; a DNAmolecule of the invention; a stop codon; and optionally a 3′ terminalregion (translation and/or transcription terminator). The signal peptideencoding region is adjacent to the polynucleotide of the invention andplaced in proper reading frame. The signal peptide-encoding region ishomologous or heterologous to the DNA molecule encoding the maturepolypeptide and is compatible with the secretion apparatus of the hostused for expression. The open reading frame constituted by the DNAmolecule of the invention, solely or together with the signal peptide,is placed under the control of the promoter so that transcription andtranslation occur in the host system. Promoters and signal peptideencoding regions are widely known and available to those skilled in theart and include, for example, the promoter of Salmonella typhimurium(and derivatives) that is inducible by arabinose (promoter araB) and isfunctional in Gram-negative bacteria such as E. coli (as described inU.S. Pat. No. 5,028,530 and in Cagnon et al., (Cagnon et al., ProteinEngineering (1991) 4(7):843)); the promoter of the gene of bacteriophageT7 encoding RNA polymerase, that is functional in a number of E. colistrains expressing T7 polymerase (described in U.S. Patent No.4,952,496); OspA lipidation signal peptide ; and RlpB lipidation signalpeptide (Takase et al., J. Bact. (1987) 169:5692).

[0091] The expression cassette is typically part of an expressionvector, which is selected for its ability to replicate in the chosenexpression system. Expression vectors (e.g., plasmids or viral vectors)can be chosen, for example, from those described in Pouwels et al.(Cloning Vectors: A Laboratory Manual 1985, Supp. 1987). Suitableexpression vectors can be purchased from various commercial sources.

[0092] Methods for transforming/transfecting host cells with expressionvectors are well-known in the art and depend on the host system selectedas described in Ausubel et al., (Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons Inc., 1994).

[0093] Upon expression, a recombinant polypeptide of the invention (or apolypeptide derivative) is produced and remains in the intracellularcompartment, is secreted/excreted in the extracellular medium or in theperiplasmic space, or is embedded in the cellular membrane. Thepolypeptide is recovered in a substantially purified form from the cellextract or from the supernatant after centrifugation of the recombinantcell culture. Typically, the recombinant polypeptide is purified byantibody-based affinity purification or by other well-known methods thatcan be readily adapted by a person skilled in the art, such as fusion ofthe polynucleotide encoding the polypeptide or its derivative to a smallaffinity binding domain. Antibodies useful for purifying byimmunoaffinity the polypeptides of the invention are obtained asdescribed below.

[0094] A polynucleotide of the invention can also be useful as avaccine. There are two major routes, either using a viral or bacterialhost as gene delivery vehicle (live vaccine vector) or administering thegene in a free form, e.g., inserted into a plasmid. Therapeutic orprophylactic efficacy of a polynucleotide of the invention is evaluatedas described below.

[0095] Accordingly, a third aspect of the invention provides (i) avaccine vector such as a poxvirus, containing a DNA molecule of theinvention, placed under the control of elements required for expression;(ii) a composition of matter comprising a vaccine vector of theinvention, together with a diluent or carrier; specifically (iii) apharmaceutical composition containing a therapeutically orprophylactically effective amount of a vaccine vector of the invention;(iv) a method for inducing an immune response against Chlamydia in amammal (e.g., a human; alternatively, the method can be used inveterinary applications for treating or preventing Chlamydia infectionof animals, e.g., cats or birds), which involves administering to themammal an immunogenically effective amount of a vaccine vector of theinvention to elicit a protective or therapeutic immune response toChlamydia ; and particularly, (v) a method for preventing and/ortreating a Chlamydia (e.g., C. trachomatis, C. psittaci, C. pneumonia,C. pecorum) infection, which involves administering a prophylactic ortherapeutic amount of a vaccine vector of the invention to an infectedindividual. Additionally, the third aspect of the invention encompassesthe use of a vaccine vector of the invention in the preparation of amedicament for preventing and/or treating Chlamydia infection.

[0096] As used herein, a vaccine vector expresses one or severalpolypeptides or derivatives of the invention. The vaccine vector mayexpress additionally a cytokine, such as interleukin-2 (IL-2) orinterleukin-12 (IL-12), that enhances the immune response (adjuvanteffect). It is understood that each of the components to be expressed isplaced under the control of elements required for expression in amammalian cell.

[0097] Consistent with the third aspect of the invention is acomposition comprising several vaccine vectors, each of them capable ofexpressing a polypeptide or derivative of the invention. A compositionmay also comprise a vaccine vector capable of expressing an additionalChlamydia antigen, or a subunit, fragment, homolog, mutant, orderivative thereof; optionally together with or a cytokine such as IL-2or IL-12.

[0098] Vaccination methods for treating or preventing infection in amammal comprises use of a vaccine vector of the invention to beadministered by any conventional route, particularly to a mucosal (e.g.,ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal,vaginal, or urinary tract) surface or via the parenteral (e.g.,subcutaneous, intradermal, intramuscular, intravenous, orintraperitoneal) route. Preferred routes depend upon the choice of thevaccine vector. Treatment may be effected in a single dose or repeatedat intervals. The appropriate dosage depends on various parametersunderstood by skilled artisans such as the vaccine vector itself, theroute of administration or the condition of the mammal to be vaccinated(weight, age and the like).

[0099] Live vaccine vectors available in the art include viral vectorssuch as adenoviruses and poxviruses as well as bacterial vectors, e.g.,Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilié deCalmette-Guérin (BCG), and Streptococcus.

[0100] An example of an adenovirus vector, as well as a method forconstructing an adenovirus vector capable of expressing a DNA moleculeof the invention, are described in U.S. Pat. No. 4,920,209. Poxvirusvectors include vaccinia and canary pox virus, described in U.S. Pat.No. 4,722,848 and U.S. Pat. No. 5,364,773, respectively. (Also see,e.g., Tartaglia et al., Virology (1992) 188:217) for a description of avaccinia virus vector and Taylor et al, Vaccine (1995) 13:539 for areference of a canary pox.) Poxvirus vectors capable of expressing apolynucleotide of the invention are obtained by homologous recombinationas described in Kieny et al., Nature (1984) 312:163 so that thepolynucleotide of the invention is inserted in the viral genome underappropriate conditions for expression in mammalian cells. Generally, thedose of vaccine viral vector, for therapeutic or prophylactic use, canbe of from about 1×10⁴ to about 1×10¹¹, advantageously from about 1×10⁷to about 1×10¹⁰, preferably of from about 1×10⁷ to about 1×10⁹plaque-forming units per kilogram. Preferably, viral vectors areadministered parenterally; for example, in 3 doses, 4 weeks apart. It ispreferable to avoid adding a chemical adjuvant to a compositioncontaining a viral vector of the invention and thereby minimizing theimmune response to the viral vector itself.

[0101] Non-toxicogenic Vibrio cholerae mutant strains that are useful asa live oral vaccine are known. Mekalanos et al., Nature (1983) 306:551and U.S. Pat. No. 4,882,278 describe strains which have a substantialamount of the coding sequence of each of the two ctxA alleles deleted sothat no functional cholerae toxin is produced. WO 92/11354 describes astrain in which the irgA locus is inactivated by mutation; this mutationcan be combined in a single strain with ctxA mutations. wo 94/01533describes a deletion mutant lacking functional ctxA and attRS1 DNAsequences. These mutant strains are genetically engineered to expressheterologous antigens, as described in WO 94/19482. An effective vaccinedose of a Vibrio cholerae strain capable of expressing a polypeptide orpolypeptide derivative encoded by a DNA molecule of the inventioncontains about 1×10⁵ to about 1×10⁹, preferably about 1×10⁶ to about1×10⁸, viable bacteria in a volume appropriate for the selected route ofadministration. Preferred routes of administration include all mucosalroutes; most preferably, these vectors are administered intranasally ororally.

[0102] Attenuated Salmonella typhimurium strains, genetically engineeredfor recombinant expression of heterologous antigens or not, and theiruse as oral vaccines are described in Nakayama et al. (Bio/Technology(1988) 6:693) and WO 92/11361. Preferred routes of administrationinclude all mucosal routes; most preferably, these vectors areadministered intranasally or orally.

[0103] Other bacterial strains used as vaccine vectors in the context ofthe present invention are described for Shigella flexneri in High etal., EMBO (1992) 11:1991 and Sizemore et al., Science (1995) 270:299;for Streptococcus gordonii in Medaglini et al., Proc. Natl. Acad. Sci.USA (1995) 92:6868; and for Bacille Calmette Guerin in Flynn J. L.,Cell. Mol. Biol. (1994) 40 (suppl. I):31, WO 88/06626, WO 90/00594, WO91/13157, WO 92/01796, and WO 92/21376.

[0104] In bacterial vectors, the polynucleotide of the invention isinserted into the bacterial genome or remains in a free state as part ofa plasmid.

[0105] The composition comprising a vaccine bacterial vector of thepresent invention may further contain an adjuvant. A number of adjuvantsare known to those skilled in the art. Preferred adjuvants are selectedas provided below.

[0106] Accordingly, a fourth aspect of the invention provides (i) acomposition of matter comprising a polynucleotide of the invention,together with a diluent or carrier; (ii) a pharmaceutical compositioncomprising a therapeutically or prophylactically effective amount of apolynucleotide of the invention; (iii) a method for inducing an immuneresponse against Chlamydia in a mammal by administration of animmunogenically effective amount of a polynucleotide of the invention toelicit a protective immune response to Chlamydia; and particularly, (iv)a method for preventing and/or treating a Chlamydia (e.g., C.trachomatis, C. psittaci, C. pneumoniae, or C. pecorum) infection, byadministering a prophylactic or therapeutic amount of a polynucleotideof the invention to an infected individual. Additionally, the fourthaspect of the invention encompasses the use of a polynucleotide of theinvention in the preparation of a medicament for preventing and/ortreating Chlamydia infection. A preferred use includes the use of a DNAmolecule placed under conditions for expression in a mammalian cell,especially in a plasmid that is unable to replicate in mammalian cellsand to substantially integrate in a mammalian genome.

[0107] Use of the polynucleotides of the invention include theiradministration to a mammal as a vaccine, for therapeutic or prophylacticpurposes. Such polynucleotides are used in the form of DNA as part of aplasmid that is unable to replicate in a mammalian cell and unable tointegrate into the mammalian genome. Typically, such a DNA molecule isplaced under the control of a promoter suitable for expression in amammalian cell. The promoter functions either ubiquitously ortissue-specifically. Examples of non-tissue specific promoters includethe early Cytomegalovirus (CMV) promoter (described in U.S. Pat. No.4,168,062) and the Rous Sarcoma Virus promoter (described in Norton &Coffin, Molec. Cell Biol. (1985) 5:281). An example of a tissue-specificpromoter is the desmin promoter which drives expression in muscle cells(Li et al., Gene (1989) 78:243, Li & Paulin, J. Biol. Chem. (1991)266:6562 and Li & Paulin, J. Biol. Chem. (1993) 268:10403). Use ofpromoters is well-known to those skilled in the art. Useful vectors aredescribed in numerous publications, specifically WO 94/21797 andHartikka et al., Human Gene Therapy (1996) 7:1205.

[0108] Polynucleotides of the invention which are used as vaccinesencode either a precursor or a mature form of the correspondingpolypeptide. In the precursor form, the signal peptide is eitherhomologous or heterologous. In the latter case, a eucaryotic leadersequence such as the leader sequence of the tissue-type plasminogenfactor (tPA) is preferred.

[0109] As used herein, a composition of the invention contains one orseveral polynucleotides with optionally at least one additionalpolynucleotide encoding another Chlamydia antigen such as urease subunitA, B, or both, or a fragment, derivative, mutant, or analog thereof. Thecomposition may also contain an additional polynucleotide encoding acytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12) so thatthe immune response is enhanced. These additional polynucleotides areplaced under appropriate control for expression. Advantageously, DNAmolecules of the invention and/or additional DNA molecules to beincluded in the same composition, are present in the same plasmid.

[0110] Standard techniques of molecular biology for preparing andpurifying polynucleotides are used in the preparation of polynucleotidetherapeutics of the invention. For use as a vaccine, a polynucleotide ofthe invention is formulated according to various methods outlined below.

[0111] One method utililizes the polynucleotide in a naked form, free ofany delivery vehicles. Such a polynucleotide is simply diluted in aphysiologically acceptable solution such as sterile saline or sterilebuffered saline, with or without a carrier. When present, the carrierpreferably is isotonic, hypotonic, or weakly hypertonic, and has arelatively low ionic strength, such as provided by a sucrose solution,e.g., a solution containing 20% sucrose.

[0112] An alternative method utilizes the polynucleotide in associationwith agents that assist in cellular uptake. Examples of such agents are(i) chemicals that modify cellular permeability, such as bupivacaine(see, e.g., WO 94/16737), (ii) liposomes for encapsulation of thepolynucleotide, or (iii) cationic lipids or silica, gold, or tungstenmicroparticles which associate themselves with the polynucleotides.

[0113] Anionic and neutral liposomes are well-known in the art (see,e.g., Liposomes: A Practical Approach, RPC New Ed, IRL press (1990), fora detailed description of methods for making liposomes) and are usefulfor delivering a large range of products, including polynucleotides.

[0114] Cationic lipids are also known in the art and are commonly usedfor gene delivery. Such lipids include Lipofectin™ also known as DOTMA(N-[1- (2,3 -dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP(1,2-bis (oleyloxy) -3-(trimethylammonio)propane), DDAB(dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycylspermine) and cholesterol derivatives such as DC-Chol (3beta-(N-(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol). Adescription of these cationic lipids can be found in EP 187,702, WO90/11092, U.S. Pat. No. 5,283,185, WO 91/15501, WO 95/26356, and U.S.Pat. No. 5,527,928. Cationic lipids for gene delivery are preferablyused in association with a neutral lipid such as DOPE (dioleylphosphatidylethanolamine), as described in WO 90/11092 as an example.

[0115] Formulation containing cationic liposomes may optionally containother transfection-facilitating compounds. A number of them aredescribed in WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/02397.They include spermine derivatives useful for facilitating the transportof DNA through the nuclear membrane (see, for example, WO 93/18759) andmembrane-permeabilizing compounds such as GALA, Gramicidine S, andcationic bile salts (see, for example, WO 93/19768).

[0116] Gold or tungsten microparticles are used for gene delivery, asdescribed in WO 91/00359, WO 93/17706, and Tang et al. Nature (1992)356:152. The microparticle-coated polynucleotide is injected viaintradermal or intraepidermal routes using a needleless injection device(“gene gun”), such as those described in U.S. Pat. No. 4,945,050, U.S.Pat. No. 5,015,580, and WO 94/24263.

[0117] The amount of DNA to be used in a vaccine recipient depends,e.g., on the strength of the promoter used in the DNA construct, theimmunogenicity of the expressed gene product, the condition of themammal intended for administration (e.g., the weight, age, and generalhealth of the mammal), the mode of administration, and the type offormulation. In general, a therapeutically or prophylactically effectivedose from about 1 μg to about 1 mg, preferably, from about 10 μg toabout 800 μg and, more preferably, from about 25 μg to about 250 μg, canbe administered to human adults. The administration can be achieved in asingle dose or repeated at intervals.

[0118] The route of administration is any conventional route used in thevaccine field. As general guidance, a polynucleotide of the invention isadministered via a mucosal surface, e.g., an ocular, intranasal,pulmonary, oral, intestinal, rectal, vaginal, and urinary tract surface;or via a parenteral route, e.g., by an intravenous, subcutaneous,intraperitoneal, intradermal, intraepidermal, or intramuscular route.The choice of administration route depends on the formulation that isselected. A polynucleotide formulated in association with bupivacaine isadvantageously administered into muscles. When a neutral or anionicliposome or a cationic lipid, such as DOTMA or DC-Chol, is used, theformulation can be advantageously injected via intravenous, intranasal(aerosolization), intramuscular, intradermal, and subcutaneous routes. Apolynucleotide in a naked form can advantageously be administered viathe intramuscular, intradermal, or subcutaneous routes.

[0119] Although not absolutely required, such a composition can alsocontain an adjuvant. If so, a systemic adjuvant that does not requireconcomitant administration in order to exhibit an adjuvant effect ispreferable such as, e.g., QS21, which is described in U.S. Pat. No.5,057,546.

[0120] The sequence information provided in the present applicationenables the design of specific nucleotide probes and primers that areused for diagnostic purposes. Accordingly, a fifth aspect of theinvention provides a nucleotide probe or primer having a sequence foundin or derived by degeneracy of the genetic code from a sequence shown inSEQ ID No: 1, 3 or 5

[0121] The term “probe” as used in the present application refers to DNA(preferably single stranded) or RNA molecules (or modifications orcombinations thereof) that hybridize under the stringent conditions, asdefined above, to nucleic acid molecules having SEQ ID No: 1, 3 or 5 orto sequences homologous to SEQ ID No:l, 3 or 5, or to its complementaryor anti-sense sequence. Generally, probes are significantly shorter thanfull-length sequences. Such probes contain from about 5 to about 100,preferably from about 10 to about 80, nucleotides. In particular, probeshave sequences that are at least 75%, preferably at least 85%, morepreferably 95% homologous to a portion of SEQ ID No:l, 3 or 5 or thatare complementary to such sequences. Probes may contain modified basessuch as inosine, methyl-5-deoxycytidine, deoxyuridine,dimethylamino-5-deoxyuridine, or diamino-2, 6-purine. Sugar or phosphateresidues may also be modified or substituted. For example, a deoxyriboseresidue may be replaced by a polyamide (Nielsen et al., Science (1991)254:1497) and phosphate residues may be replaced by ester groups such asdiphosphate, alkyl, arylphosphonate and phosphorothioate esters. Inaddition, the 2′-hydroxyl group on ribonucleotides may be modified byincluding such groups as alkyl groups.

[0122] Probes of the invention are used in diagnostic tests, as captureor detection probes. Such capture probes are conventionally immobilizedon a solid support, directly or indirectly, by covalent means or bypassive adsorption. A detection probe is labeled by a detection markerselected from: radioactive isotopes, enzymes such as peroxidase,alkaline phosphatase, and enzymes able to hydrolyze a chromogenic,fluorogenic, or luminescent substrate, compounds that are chromogenic,fluorogenic, or luminescent, nucleotide base analogs, and biotin.

[0123] Probes of the invention are used in any conventionalhybridization technique, such as dot blot (Maniatis et al., MolecularCloning: A Laboratory Manual (1982) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.), Southern blot (Southern, J. Mol. Biol. (1975)98:503), northern blot (identical to Southern blot with the exceptionthat RNA is used as a target), or the sandwich technique (Dunn et al.,Cell (1977) 12:23). The latter technique involves the use of a specificcapture probe and/or a specific detection probe with nucleotidesequences that at least partially differ from each other.

[0124] A primer is a probe of usually about 10 to about 40 nucleotidesthat is used to initiate enzymatic polymerization of DNA in anamplification process (e.g., PCR), in an elongation process, or in areverse transcription method. Primers used in diagnostic methodsinvolving PCR are labeled by methods known in the art.

[0125] As described herein, the invention also encompasses (i) a reagentcomprising a probe of the invention for detecting and/or identifying thepresence of Chlamydia in a biological material; (ii) a method fordetecting and/or identifying the presence of Chlamydia in a biologicalmaterial, in which (a) a sample is recovered or derived from thebiological material, (b) DNA or RNA is extracted from the material anddenatured, and (c) exposed to a probe of the invention, for example, acapture, detection probe or both, under stringent hybridizationconditions, such that hybridization is detected; and (iii) a method fordetecting and/or identifying the presence of Chlamydia in a biologicalmaterial, in which (a) a sample is recovered or derived from thebiological material, (b) DNA is extracted therefrom, (c) the extractedDNA is primed with at least one, and preferably two, primers of theinvention and amplified by polymerase chain reaction, and (d) theamplified DNA fragment is produced.

[0126] It is apparent that disclosure of polynucleotide sequences of SEQID No: 1, 3 or 5, its homologs and partial sequences enable theircorresponding amino acid sequences. Accordingly, a sixth aspect of theinvention features a substantially purified polypeptide or polypeptidederivative having an amino acid sequence encoded by a polynucleotide ofthe invention.

[0127] A “substantially purified polypeptide” as used herein is definedas a polypeptide that is separated from the environment in which itnaturally occurs and/or that is free of the majority of the polypeptidesthat are present in the environment in which it was synthesized. Forexample, a substantially purified polypeptide is free from cytoplasmicpolypeptides. Those skilled in the art would readily understand that thepolypeptides of the invention may be purified from a natural source,i.e., a Chlamydia strain, or produced by recombinant means.

[0128] Consistent with the sixth aspect of the invention arepolypeptides, homologs or fragments which are modified or treated toenhance their immunogenicity in the target animal, in whom thepolypeptide, homolog or fragments are intended to confer protectionagainst Chlamydia. Such modifications or treatments include: amino acidsubstitutions with an amino acid derivative such as 3-methyhistidine,4-hydroxyproline, 5-hydroxylysine etc., modifications or deletions whichare carried out after preparation of the polypeptide, homolog orfragment, such as the modification of free amino, carboxyl or hydroxylside groups of the amino acids.

[0129] Identification of homologous polypeptides or polypeptidederivatives encoded by polynucleotides of the invention which havespecific antigenicity is achieved by screening for cross-reactivity withan antiserum raised against the polypeptide of reference having an aminoacid sequence of SEQ ID No: 1, 3 or 5. The procedure is as follows: amonospecific hyperimmune antiserum is raised against a purifiedreference polypeptide, a fusion polypeptide (for example, an expressionproduct of MBP, GST, or His-tag systems, the description andinstructions for use of which are contained in Invitrogen productmanuals for pcDNA3.1/Myc-His(+) A, B, and C and for the Xpress™ SystemProtein Purification), or a synthetic peptide predicted to be antigenic.Where an antiserum is raised against a fusion polypeptide, two differentfusion systems are employed. Specific antigenicity can be determinedaccording to a number of methods, including Western blot (Towbin et al.,Proc. Natl. Acad. Sci. USA (1979) 76:4350), dot blot, and ELISA, asdescribed below.

[0130] In a Western blot assay, the product to be screened, either as apurified preparation or a total E. coli extract, is submitted toSDS-Page electrophoresis as described by Laemmli (Nature (1970)227:680). After transfer to a nitrocellulose membrane, the material isfurther incubated with the monospecific hyperimmune antiserum diluted inthe range of dilutions from about 1:5 to about 1:5000, preferably fromabout 1:100 to about 1:500. Specific antigenicity is shown once a bandcorresponding to the product exhibits reactivity at any of the dilutionsin the above range.

[0131] In an ELISA assay, the product to be screened is preferably usedas the coating antigen. A purified preparation is preferred, although awhole cell extract can also be used. Briefly, about 100 μl of apreparation at about 10 μg protein/ml are distributed into wells of a96-well polycarbonate ELISA plate. The plate is incubated for 2 hours at37° C. then overnight at 4° C. The plate is washed with phosphate buffersaline (PBS) containing 0.05% Tween 20 (PBS/Tween buffer). The wells aresaturated with 250 μl PBS containing 1% bovine serum albumin (BSA) toprevent non-specific antibody binding. After 1 hour incubation at 370C.,the plate is washed with PBS/Tween buffer. The antiserum is seriallydiluted in PBS/Tween buffer containing 0.5% BSA. 100 μl of dilutions areadded per well. The plate is incubated for 90 minutes at 37° C., washedand evaluated according to standard procedures. For example, a goatanti-rabbit peroxidase conjugate is added to the wells when specificantibodies were raised in rabbits. Incubation is carried out for 90minutes at 37° C. and the plate is washed. The reaction is developedwith the appropriate substrate and the reaction is measured bycolorimetry (absorbance measured spectrophotometrically). Under theabove experimental conditions, a positive reaction is shown by O.D.values greater than a non immune control serum.

[0132] In a dot blot assay, a purified product is preferred, although awhole cell extract can also be used. Briefly, a solution of the productat about 100 μg/ml is serially two-fold diluted in 50 mM Tris-HCl (pH7.5). 100 μl of each dilution are applied to a nitrocellulose membrane0.45 μm set in a 96-well dot blot apparatus (Biorad). The buffer isremoved by applying vacuum to the system. Wells are washed by additionof 50 mM Tris-HCl (pH 7.5) and the membrane is air-dried. The membraneis saturated in blocking buffer (50 mM Tris-HCl (pH 7.5) 0.15 M NaCl, 10g/L skim milk) and incubated with an antiserum dilution from about 1:50to about 1:5000, preferably about 1:500. The reaction is revealedaccording to standard procedures. For example, a goat anti-rabbitperoxidase conjugate is added to the wells when rabbit antibodies areused. Incubation is carried out 90 minutes at 37° C. and the blot iswashed. The reaction is developed with the appropriate substrate andstopped. The reaction is measured visually by the appearance of acolored spot, e.g., by colorimetry. Under the above experimentalconditions, a positive reaction is shown once a colored spot isassociated with a dilution of at least about 1:5, preferably of at leastabout 1:500.

[0133] Therapeutic or prophylactic efficacy of a polypeptide orderivative of the invention can be evaluated as described below. Aseventh aspect of the invention provides (i) a composition of mattercomprising a polypeptide of the invention together with a diluent orcarrier; specifically (ii) a pharmaceutical composition containing atherapeutically or prophylactically effective amount of a polypeptide ofthe invention; (iii) a method for inducing an immune response againstChlamydia in a mammal, by administering to the mammal an immunogenicallyeffective amount of a polypeptide of the invention to elicit aprotective immune response to Chlamydia; and particularly, (iv) a methodfor preventing and/or treating a Chlamydia (e.g., C. trachomatis. C.psittaci, C. pneumoniae. or C. pecorum) infection, by administering aprophylactic or therapeutic amount of a polypeptide of the invention toan infected individual. Additionally, the seventh aspect of theinvention encompasses the use of a polypeptide of the invention in thepreparation of a medicament for preventing and/or treating Chlamydiainfection.

[0134] As used herein, the immunogenic compositions of the invention areadministered by conventional routes known the vaccine field, inparticular to a mucosal (e.g., ocular, intranasal, pulmonary, oral,gastric, intestinal, rectal, vaginal, or urinary tract) surface or viathe parenteral (e.g., subcutaneous, intradermal, intramuscular,intravenous, or intraperitoneal) route. The choice of administrationroute depends upon a number of parameters, such as the adjuvantassociated with the polypeptide. If a mucosal adjuvant is used, theintranasal or oral route is preferred. If a lipid formulation or analuminum compound is used, the parenteral route is preferred with thesub-cutaneous or intramuscular route being most preferred. The choicealso depends upon the nature of the vaccine agent. For example, apolypeptide of the invention fused to CTB or LTB is best administered toa mucosal surface.

[0135] As used herein, the composition of the invention contains one orseveral polypeptides or derivatives of the invention. The compositionoptionally contains at least one additional Chlamydia antigen, or asubunit, fragment, homolog, mutant, or derivative thereof.

[0136] For use in a composition of the invention, a polypeptide orderivative thereof is formulated into or with liposomes, preferablyneutral or anionic liposomes, microspheres, ISCOMS, orvirus-like-particles (VLPs) to facilitate delivery and/or enhance theimmune response. These compounds are readily available to one skilled inthe art; for example, see Liposomes: A Practical Approach, RCP New Ed,IRL press (1990).

[0137] Adjuvants other than liposomes and the like are also used and areknown in the art. Adjuvants may protect the antigen from rapid dispersalby sequestering it in a local deposit, or they may contain substancesthat stimulate the host to secrete factors that are chemotactic formacrophages and other components of the immune system. An appropriateselection can conventionally be made by those skilled in the art, forexample, from those described below (under the eleventh aspect of theinvention).

[0138] Treatment is achieved in a single dose or repeated as necessaryat intervals, as can be determined readily by one skilled in the art.For example, a priming dose is followed by three booster doses at weeklyor monthly intervals. An appropriate dose depends on various parametersincluding the recipient (e.g., adult or infant), the particular vaccineantigen, the route and frequency of administration, the presence/absenceor type of adjuvant, and the desired effect (e.g., protection and/ortreatment), as can be determined by one skilled in the art. In general,a vaccine antigen of the invention is administered by a mucosal route inan amount from about 10 μg to about 500 mg, preferably from about 1 mgto about 200 mg. For the parenteral route of administration, the doseusually does not exceed about 1 mg, preferably about 100 μg.

[0139] When used as vaccine agents, polynucleotides and polypeptides ofthe invention may be used sequentially as part of a multistepimmunization process. For example, a mammal is initially primed with avaccine vector of the invention such as a pox virus, e.g., via theparenteral route, and then boosted twice with the polypeptide encoded bythe vaccine vector, e.g., via the mucosal route. In another example,liposomes associated with a polypeptide or derivative of the inventionis also used for priming, with boosting being carried out mucosallyusing a soluble polypeptide or derivative of the invention incombination with a mucosal adjuvant (e.g., LT).

[0140] A polypeptide derivative of the invention is also used inaccordance with the seventh aspect as a diagnostic reagent for detectingthe presence of anti-Chlamydia antibodies, e.g., in a blood sample. Suchpolypeptides are about 5 to about 80, preferably about 10 to about 50amino acids in length. They are either labeled or unlabeled, dependingupon the diagnostic method. Diagnostic methods involving such a reagentare described below.

[0141] Upon expression of a DNA molecule of the invention, a polypeptideor polypeptide derivative is produced and purified using knownlaboratory techniques. As described above, the polypeptide orpolypeptide derivative may be produced as a fusion protein containing afused tail that facilitates purification. The fusion product is used toimmunize a small mammal, e.g., a mouse or a rabbit, in order to raiseantibodies against the polypeptide or polypeptide derivative(monospecific antibodies). Accordingly, an eighth aspect of theinvention provides a monospecific antibody that binds to a polypeptideor polypeptide derivative of the invention.

[0142] By “monospecific antibody” is meant an antibody that is capableof reacting with a unique naturally-occurring Chlamydia polypeptide. Anantibody of the invention is either polyclonal or monoclonal.Monospecific antibodies may be recombinant, e.g., chimeric (e.g.,constituted by a variable region of murine origin associated with ahuman constant region), humanized (a human immunoglobulin constantbackbone together with hypervariable region of animal, e.g., murine,origin), and/or single chain. Both polyclonal and monospecificantibodies may also be in the form of immunoglobulin fragments, e.g.,F(ab)′2 or Fab fragments. The antibodies of the invention are of anyisotype, e.g., IgG or IgA, and polyclonal antibodies are of a singleisotype or a mixture of isotypes.

[0143] Antibodies against the polypeptides, homologs or fragments of thepresent invention are generated by immunization of a mammal with acomposition comprising said polypeptide, homolog or fragment. Suchantibodies may be polyclonal or monoclonal. Methods to producepolyclonal or monoclonal antibodies are well known in the art. For areview, see “Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Eds. E. Harlow and D. Lane (1988), and D. E. Yelton et al.,1981. Ann. Rev. Biochem. 50:657-680. For monoclonal antibodies, seeKohler & Milstein (1975) Nature 256:495-497.

[0144] The antibodies of the invention, which are raised to apolypeptide or polypeptide derivative of the invention, are produced andidentified using standard immunological assays, e.g., Western blotanalysis, dot blot assay, or ELISA (see, e.g., Coligan et al., CurrentProtocols in Immunology (1994) John Wiley & Sons, Inc., New York, N.Y.).The antibodies are used in diagnostic methods to detect the presence ofa Chlamydia antigen in a sample, such as a biological sample. Theantibodies are also used in affinity chromatography for purifying apolypeptide or polypeptide derivative of the invention. As is discussedfurther below, such antibodies may be used in prophylactic andtherapeutic passive immunization methods.

[0145] Accordingly, a ninth aspect of the invention provides (i) areagent for detecting the presence of Chlamydia in a biological samplethat contains an antibody, polypeptide, or polypeptide derivative of theinvention; and (ii) a diagnostic method for detecting the presence ofChlamydia in a biological sample, by contacting the biological samplewith an antibody, a polypeptide, or a polypeptide derivative of theinvention, such that an immune complex is formed, and by detecting suchcomplex to indicate the presence of Chlamydia in the sample or theorganism from which the sample is derived.

[0146] Those skilled in the art will readily understand that the immunecomplex is formed between a component of the sample and the antibody,polypeptide, or polypeptide derivative, whichever is used, and that anyunbound material is removed prior to detecting the complex. It isunderstood that a polypeptide reagent is useful for detecting thepresence of anti-Chlamydia antibodies in a sample, e.g., a blood sample,while an antibody of the invention is used for screening a sample, suchas a gastric extract or biopsy, for the presence of Chlamydiapolypeptides.

[0147] For diagnostic applications, the reagent (i.e., the antibody,polypeptide, or polypeptide derivative of the invention) is either in afree state or immobilized on a solid support, such as a tube, a bead, orany other conventional support used in the field. Immobilization isachieved using direct or indirect means. Direct means include passiveadsorption (non-covalent binding) or covalent binding between thesupport and the reagent. By “indirect means” is meant that ananti-reagent compound that interacts with a reagent is first attached tothe solid support. For example, if a polypeptide reagent is used, anantibody that binds to it can serve as an anti-reagent, provided that itbinds to an epitope that is not involved in the recognition ofantibodies in biological samples. Indirect means may also employ aligand-receptor system, for example, where a molecule such as a vitaminis grafted onto the polypeptide reagent and the corresponding receptorimmobilized on the solid phase. This is illustrated by thebiotin-streptavidin system. Alternatively, a peptide tail is addedchemically or by genetic engineering to the reagent and the grafted orfused product immobilized by passive adsorption or covalent linkage ofthe peptide tail.

[0148] Such diagnostic agents may be included in a kit which alsocomprises instructions for use. The reagent is labeled with a detectionmeans which allows for the detection of the reagent when it is bound toits target. The detection means may be a fluorescent agent such asfluorescein isocyanate or fluorescein isothiocyanate, or an enzyme suchas horse radish peroxidase or luciferase or alkaline phosphatase, or aradioactive element such as ¹²⁵I or ⁵¹Cr.

[0149] Accordingly, a tenth aspect of the invention provides a processfor purifying, from a biological sample, a polypeptide or polypeptidederivative of the invention, which involves carrying out antibody-basedaffinity chromatography with the biological sample, wherein the antibodyis a monospecific antibody of the invention.

[0150] For use in a purification process of the invention, the antibodyis either polyclonal or monospecific, and preferably is of the IgG type.Purified IgGs is prepared from an antiserum using standard methods (see,e.g., Coligan et al., Current Protocols in Immunology (1994)John Wiley &Sons, Inc., New York, N.Y.). Conventional chromatography supports, aswell as standard methods for grafting antibodies, are described in,e.g., Antibodies: A Laboratory Manual, D. Lane, E. Harlow, Eds. (1988)and outlined below.

[0151] Briefly, a biological sample, such as an C. pneumoniae extractpreferably in a buffer solution, is applied to a chromatographymaterial, preferably equilibrated with the buffer used to dilute thebiological sample so that the polypeptide or polypeptide derivative ofthe invention (i.e., the antigen) is allowed to adsorb onto thematerial. The chromatography material, such as a gel or a resin coupledto an antibody of the invention, is in either a batch form or a column.The unbound components are washed off and the antigen is then elutedwith an appropriate elution buffer, such as a glycine buffer or a buffercontaining a chaotropic agent, e.g., guanidine HCl, or high saltconcentration (e.g., 3 M MgCl₂). Eluted fractions are recovered and thepresence of the antigen is detected, e.g., by measuring the absorbanceat 280 nm.

[0152] An eleventh aspect of the invention provides (i) a composition ofmatter comprising a monospecific antibody of the invention, togetherwith a diluent or carrier; (ii) a pharmaceutical composition comprisinga therapeutically or prophylactically effective amount of a monospecificantibody of the invention, and (iii) a method for treating or preventinga Chlamydia (e.g., C. trachomatis, C. psittaci, C. pneumoniae or C.pecorum) infection, by administering a therapeutic or prophylacticamount of a monospecific antibody of the invention to an infectedindividual. Additionally, the eleventh aspect of the inventionencompasses the use of a monospecific antibody of the invention in thepreparation of a medicament for treating or preventing Chlamydiainfection.

[0153] The monospecific antibody is either polyclonal or monoclonal,preferably of the IgA isotype (predominantly). In passive immunization,the antibody is administered to a mucosal surface of a mammal, e.g., thegastric mucosa, e.g., orally or intragastrically, advantageously, in thepresence of a bicarbonate buffer. Alternatively, systemicadministration, not requiring a bicarbonate buffer, is carried out. Amonospecific antibody of the invention is administered as a singleactive component or as a mixture with at least one monospecific antibodyspecific for a different Chlamydia polypeptide. The amount of antibodyand the particular regimen used are readily determined by one skilled inthe art. For example, daily administration of about 100 to 1,000 mg ofantibodies over one week, or three doses per day of about 100 to 1,000mg of antibodies over two or three days, are effective regimens for mostpurposes.

[0154] Therapeutic or prophylactic efficacy are evaluated using standardmethods in the art, e.g., by measuring induction of a mucosal immuneresponse or induction of protective and/or therapeutic immunity, using,e.g., the C. pneumoniae mouse model. Those skilled in the art willreadily recognize that the C. pneumoniae strain of the model may bereplaced with another Chlamydia strain. For example, the efficacy of DNAmolecules and polypeptides from C. pneumoniae is preferably evaluated ina mouse model using C. pneumoniae strain. Protection is determined bycomparing the degree of Chlamydia infection to that of a control group.Protection is shown when infection is reduced by comparison to thecontrol group. Such an evaluation is made for polynucleotides, vaccinevectors, polypeptides and derivatives thereof, as well as antibodies ofthe invention.

[0155] Adjuvants useful in any of the vaccine compositions describedabove are as follows.

[0156] Adjuvants for parenteral administration include aluminumcompounds, such as aluminum hydroxide, aluminum phosphate, and aluminumhydroxy phosphate. The antigen is precipitated with, or adsorbed onto,the aluminum compound according to standard protocols. Other adjuvants,such as RIBI (ImmunoChem, Hamilton, Mont.), are used in parenteraladministration.

[0157] Adjuvants for mucosal administration include bacterial toxins,e.g., the cholera toxin (CT), the E. coli heat-labile toxin (LT), theClostridium difficile toxin A and the pertussis toxin (PT), orcombinations, subunits, toxoids, or mutants thereof such as a purifiedpreparation of native cholera toxin subunit B (CTB). Fragments,homologs, derivatives, and fusions to any of these toxins are alsosuitable, provided that they retain adjuvant activity. Preferably, amutant having reduced toxicity is used. Suitable mutants are described,e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/06627 (Arg-192-Gly LTmutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant).Additional LT mutants that are used in the methods and compositions ofthe invention include, e.g., Ser-63-Lys, Ala-69Gly, Glu-110-Asp, andGlu-112-Asp mutants. Other adjuvants, such as a bacterial monophosphoryllipid A (MPLA) of, e.g., E. coli, Salmonella minnesota, Salmonellatyphimurium, or Shigella flexneri; saponins, or polylactide glycolide(PLGA) microspheres, is also be used in mucosal administration.

[0158] Adjuvants useful for both mucosal and parenteral administrationsinclude polyphosphazene (WO 95/02415), DC-chol (3 b-(N-(N′,N′-dimethylaminomethane)-carbamoyl) cholesterol; U.S. Pat. No. 5,283,185 and wO96/14831) and QS-21 (WO 88/09336).

[0159] Any pharmaceutical composition of the invention containing apolynucleotide, a polypeptide, a polypeptide derivative, or an antibodyof the invention, is manufactured in a conventional manner. Inparticular, it is formulated with a pharmaceutically acceptable diluentor carrier, e.g., water or a saline solution such as phosphate buffersaline. In general, a diluent or carrier is selected on the basis of themode and route of administration, and standard pharmaceutical practice.Suitable pharmaceutical carriers or diluents, as well as pharmaceuticalnecessities for their use in pharmaceutical formulations, are describedin Remington's Pharmaceutical Sciences, a standard reference text inthis field and in the USP/NF.

[0160] The invention also includes methods in which Chlamydia infectionare treated by oral administration of a Chlamydia polypeptide of theinvention and a mucosal adjuvant, in combination with an antibiotic, anantacid, sucralfate, or a combination thereof. Examples of suchcompounds that can be administered with the vaccine antigen and theadjuvant are antibiotics, including, e.g., macrolides, tetracyclines,and derivatives thereof (specific examples of antibiotics that can beused include azithromycin or doxicyclin or immunomodulators such ascytokines or steroids). In addition, compounds containing more than oneof the above-listed components coupled together, are used. The inventionalso includes compositions for carrying out these methods, i.e.,compositions containing a Chlamydia antigen (or antigens) of theinvention, an adjuvant, and one or more of the above-listed compounds,in a pharmaceutically acceptable carrier or diluent.

[0161] It has recently been shown that the 9 kDa cysteine rich membraneprotein contains a sequence cross-reactive with the murine alpha-myosinheavy chain epitope M7A-alpha, an epitope conserved in humans (Bachmaieret al., Science (1999) 283:1335). This cross-reactivity is proposed tocontribute to the development of cardiovascular disease, so it may bebeneficial to remove this epitope, and any other epitopes cross-reactivewith human antigens, from the protein if it is to be used as a vaccine.Accordingly, a further embodiment of the present invention includes themodification of the coding sequence, for example, by deletion orsubstitution of the nucleotides encoding the epitope frompolynucleotides encoding the protein, as to improve the efficacy andsafety of the protein as a vaccine. A similar approach may beappropriate for any protective antigen found to have unwanted homologiesor cross-reactivities with human antigens.

[0162] Amounts of the above-listed compounds used in the methods andcompositions of the invention are readily determined by one skilled inthe art. Treatment/immunization schedules are also known and readilydesigned by one skilled in the art. For example, the non-vaccinecomponents can be administered on days 1-14, and the vaccine antigen+adjuvant can be administered on days 7, 14, 21, and 28.

EXAMPLES

[0163] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific examples. These examples are described solely forpurposes of illustration and are not intended to limit the scope of theinvention. Changes in form and substitution of equivalents arecontemplated as circumstances may suggest or render expedient. Althoughspecific terms have been employed herein, such terms are intended in adescriptive sense and not for purposes of limitation.

Example 1

[0164] This example illustrates the preparation of a plasmid vectorpCACPNM555a containing the full length 76 kDa protein gene.

[0165] The full-length 76 kDa protein gene was amplified from Chlamydiapneumoniae genomic DNA by polymerase chain reaction (PCR) using a 5′primer (5′ ATAAGAATGCGGCCGC CACCATGGTTAATCCTATTGGTCCAGG 3′) (SEQ IDNo:9) and a 3′ primer (5′ GCGCCGGATCC CTTGGAGATAACCAGAATATAGAG 3′) (SEQID No:10). The 5′ primer contains a Not I restriction site, a ribosomebinding site, an initiation codon and a sequence close to the 5′ end ofthe full-length 76 kDa protein coding sequence. The 3′primer includesthe sequence encoding the C-terminal sequence of the 76 kDa protein anda Bam HI restriction site. The stop codon was excluded and an additionalnucleotide was inserted to obtain an in-frame fusion with the Histidinetag.

[0166] After amplification, the PCR fragment was purified usingQIAquick™ PCR purification kit (Qiagen) and then digested with Not I andBam HI and cloned into the pCA-Myc-His eukaryotic expression vectordescribe in Example 2 (FIG. 4) with transcription under control of thehuman CMV promoter.

Example 2

[0167] This example illustrates the preparation of the eukaryoticexpression vector pCA/Myc-His.

[0168] Plasmid pcDNA3.1(−)Myc-His C (Invitrogen) was restricted with SpeI and Bam HI to remove the CMV promoter and the remaining vectorfragment was isolated. The CMV promoter and intron A from plasmidVR-1012 (Vical) was isolated on a Spe I/Bam HI fragment. The fragmentswere ligated together to produce plasmid pCA/Myc-His. The Not I/Bam HIrestricted PCR fragment containing the full-length 76 kDa protein genewas ligated into the Not I and Bam HI restricted plasmid pCA/Myc-His toproduce plasmid pCACPNM555a (FIG. 4).

[0169] The resulting plasmid, pCACPNM555a, was transferred byelectroporation into E. coli XL-1 blue (Stratagene) which was grown inLB broth containing 50 μg/ml of carbenicillin. The plasmid was isolatedby Endo Free Plasmid Giga Kit™ (Qiagen) large scale DNA purificationsystem. DNA concentration was determined by absorbance at 260 nm and theplasmid was verified after gel electrophoresis and Ethidium bromidestaining and comparison to molecular weight standards. The 5′ and 3′ends of the gene were verified by sequencing using a LiCor model 4000 LDNA sequencer and IRD-800 labelled primers.

Example 3

[0170] This example illustrates the immunization of mice to achieveprotection against an intranasal challenge of C. pneumoniae.

[0171] It has been previously demonstrated (Yang et. al., 1993) thatmice are susceptible to intranasal infection with different isolates ofC. pneumoniae. Strain AR-39 (Grayston, 1989) was used in Balb/c mice asa challenge infection model to examine the capacity of Chlamydia geneproducts delivered as naked DNA to elicit a protective response againsta sublethal C. pneumoniae lung infection. Protective immunity is definedas an accelerated clearance of pulmonary infection.

[0172] Groups of 7 to 9 week old male Balb/c mice (7 to 10 per group)were immunized intramuscularly (i.m.) plus intranasally (i.n.) withplasmid DNA containing the coding sequence of C.pneumoniae full-length76kDa protein as described in Examples 1 and 2. Saline or the plasmidvector lacking an inserted Chlamydial gene was given to groups ofcontrol animals.

[0173] For i.m. immunization alternate left and right quadriceps wereinjected with 100 μg of DNA in 50 μl of PBS on three occasions at 0, 3and 6 weeks. For i.n. immunization, anaesthetized mice aspirated 50 μlof PBS containing 50 μg DNA on three occasions at 0, 3 and 6 weeks. Atweek 8, immunized mice were inoculated i.n. with 5×10⁵ IFU of C.pneumoniae, strain AR39 in 100 μl of SPG buffer to test their ability tolimit the growth of a sublethal C. pneumoniae challenge.

[0174] Lungs were taken from mice at days 5 and 9 post-challenge andimmediately homogenised in SPG buffer (7.5% sucrose, 5 mM glutamate,12.5 mM phosphate pH7.5). The homogenate was stored frozen at −70° C.until assay. Dilutions of the homogenate were assayed for the presenceof infectious Chlamydia by inoculation onto monolayers of susceptiblecells. The inoculum was centrifuged onto the cells at 3000 rpm for 1hour, then the cells were incubated for three days at 35° C. in thepresence of 1 μg/ml cycloheximide. After incubation the monolayers werefixed with formalin and methanol then immunoperoxidase stained for thepresence of Chlamydial inclusions using convalescent sera from rabbitsinfected with C.pneumoniae and metal-enhanced DAB as a peroxidasesubstrate.

[0175]FIG. 7 and Table 1 show that mice immunized i.n. and i.m. withpCACPNM555a had Chlamydial lung titers less than 30,000 IFU/lung (mean23,550) in 5 of 6 cases at day 9 whereas the range of values for controlmice sham immunized with saline were 20,800 to 323,300 IFU/lung (mean206,375) for (Table 1). DNA immunisation per se was not responsible forthe observed protective effect since two other plasmid DNA constructs,pCACPNM806 and pCACPNM760, failed to protect, with lung titers inimmunised mice similar to those obtained for saline-immunized controlmice. The constructs pCACPNM806 and pCACPNM760 are identical topCACPNM555a except that the nucleotide sequence encoding the full-length76kDa proteinis replaced with C. pneumoniae nucleotide sequencesencoding an unrelated sequence. TABLE 1 BACTERIAL LOAD (INCLUSIONFORMING UNITS PER LUNG) IN THE LUNGS OF BALB/C MICE IMMUNIZED WITHVARIOUS DNA IMMUNIZATION CONSTRUCTS IMMUNIZING CONSTRUCT SalinepCACPNM806 pCACPNN760 pCACPNM555a MOUSE Day 9 Day 9 Day 9 Day 9 1 22590036700 140300 27300 2 20800 238700 128400 15200 3 286100 52300 8870034600 4 106700 109600 25600 20500 5 323300 290000 37200 22000 6 144300298800 5900 21700 7 261700 8 282200 MEAN 206375 171016.667 71016.666723550 SD 105183.9 119141.32 56306.57 6648.53 Wilcoxon p 0.8518 0.02930.008

Example 4

[0176] This example illustrates the preparation of a plasmid vectorpCAI555 containing a 5′-truncated 76 kDa protein gene.

[0177] The 5′ truncated 76 kDa protein gene was amplified from Chlamydiapneumoniae genomic DNA by polymerase chain reaction (PCR) using a 5′primer (5′ ATAAGAATGCGGCCGC CACCATGAGTCTGGCAGATAAGCTGGG 3′) (SEQ ID No:7and a 3′ primer (5′ GCGCCGGATCC CTTGGAGATAACCAGAATATA 3′) (SEQ ID No:8).The 5′ primer contains a Not I restriction site, a ribosome bindingsite, an initiation codon and a sequence at the second Met codon of the76 kDa protein coding sequence. The 3′ primer includes the sequenceencoding the C-terminal sequence of the 3′ 76 kDa protein and a Bam HIrestriction site. The stop codon was excluded and an additionalnucleotide was inserted to obtain an in-frame fusion with the Histidinetag.

[0178] After amplification, the PCR fragment was purified usingQIAquick™ PCR purification kit (Qiagen) and then digested with Not I andBam HI and cloned into the pCA-Myc-His eukaryotic expression vectordescribe in Example 5 (FIG. 5) with transcription under control of thehuman CMV promoter.

Example 5

[0179] This example illustrates the preparation of the eukaryoticexpression vector pCA/Myc-His.

[0180] Plasmid pcDNA3.1(−)Myc-His C (Invitrogen) was restricted with SpeI and Dam HI to remove the CMV promoter and the remaining vectorfragment was isolated. The CMV promoter and intron A from plasmidVR-1012 (Vical) was isolated on a Spe I/Dam HI fragment. The fragmentswere ligated together to produce plasmid pCA/Myc-His. The Not I/Bam HIrestricted PCR fragment containing the 5′ truncated 76 kDa protein genewas ligated into the Not I and Bam HI restricted plasmid pCA/Myc-His toproduce plasmid pCAI555 (FIG. 5).

[0181] The resulting plasmid, pCAI555, was transferred byelectroporation into E. coli XL-1 blue (Stratagene) which was grown inLB broth containing 50 μg/ml of carbenicillin. The plasmid was isolatedby Endo Free Plasmid Giga Kit™ (Qiagen) large scale DNA purificationsystem. DNA concentration was determined by absorbance at 260 nm and theplasmid was verified after gel electrophoresis and Ethidium bromidestaining and comparison to molecular weight standards. The 5′ and 3′ends of the gene were verified by sequencing using a LiCor model 4000 LDNA sequencer and IRD-800 labelled primers.

Example 6

[0182] This Example illustrates the immunization of mice to achieveprotection against an intranasal challenge of C. pneumoniae. Theprocedures are described in Example 3 above, except that the DNA plasmidused for immunization contains the coding sequence of C. pneumoniae5′-truncated 76 kDa protein, as described in Examples 4 and 5.

[0183]FIG. 8 and Table 2 show that mice immunized i.n. and i.m. withpCAI555 had Chlamydial lung titers less than 13000 IFU/lung (mean 6050)in 6 of 6 cases at day 9 whereas the range of values for control micesham immunized with saline were 106,100 IFU/lung (mean 39,625) for(Table 2). DNA immunisation per se was not responsible for the observedprotective effect since two other plasmid DNA constructs, pCAI116 andpCAI178, failed to protect, with lung titers in immunised mice similarto those obtained for saline-immunized control mice. The constructspCAI116 and pCAI178 are identical to pCAI555 except that the nucleotidesequence encoding the 5′-truncated 76 kDa protein is replaced with aC.pneumoniae nucleotide sequence encoding an unprotective sequence andthe nucleoside 5′-diphosphate phosphotransferase protein. TABLE 2BACTERIAL LOAD (INCLUSION FORMING UNITS PER LUNG) IN THE LUNGS OF BALB/CMICE IMMUNIZED WITH VARIOUS DNA IMMUNIZATION CONSTRUCTS IMMUNIZINGCONSTRUCT Saline pCAI116 pCAI178 pCAI555 MOUSE Day 9 Day 9 Day 9 Day 9 11700 47700 80600 6100 2 36200 12600 31900 10700 3 106100 28600 30600 5004 33500 17700 6500 5100 5 70400 77300 53000 1100 6 48700 17600 7950012800 7 600 8 19800 9 29500 10 100000 11 15000 12 56600 13 60300 1488800 15 30400 16 69300 17 47500 18 96500 19 30200 20 84800 21 3800 2265900 23 33000 MEAN 49069.57 33583.33 47016.67 6050 SD 32120.48 24832.6729524.32 4967.80

Example 7

[0184] This example illustrates the preparation of a plasmid vectorpCAD76kDa containing a 3′-truncated 76 kDa protein gene.

[0185] The 3′-truncated 76 kDa protein gene was amplified from Chlamydiapneumoniae genomic DNA by polymerase chain reaction (PCR) using a 5′primer (5′ GCTCTAGA CCGCCATGACAAAAAAACATTATGCTTGGG 3′) (SEQ ID No:9 )and a 3′ primer (5′ CGGGATCC ATAGAACTTGCTGCAGCGGG 3′) (SEQ ID No:10).The 5′ primer contains a Xba I restriction site, a ribosome bindingsite, an initiation codon and a sequence 765 bp upstream of the 5′ endof the 76 kDa protein coding sequence. The 3′ primer includes a 21 bpthe sequence downstream of codon 452 of the 76 kDa protein and a Bam HIrestriction site. An additional nucleotide was inserted to obtain anin-frame fusion with the Histidine tag. Note that inclusion of the 765bp 5′ region and the 21 bp 3′ regions were inadvertent. These sequencesare not part of the 76 kDa protein gene. Nevertheless, immunoprotectionwas achieved using this sequence (Example 6).

[0186] After amplification, the PCR fragment was purified usingQIAquick™ PCR purification kit (Qiagen) and then digested with Xba I andBam HI and cloned into the pCA-Myc-His eukaryotic expression vectordescribe in Example 8 (FIG. 6) with transcription under control of thehuman CMV promoter.

Example 8

[0187] This Example illustrates the preparation of the eukaryoticexpression vector pCA/Myc-His.

[0188] Plasmid pcDNA3.1(−)Myc-His C (Invitrogen) was restricted with SpeI and Bam HI to remove the CMV promoter and the remaining vectorfragment was isolated. The CMV promoter and intron A from plasmidVR-1012 (Vical) was isolated on a Spe I/Bam HI fragment. The fragmentswere ligated together to produce plasmid pCA/Myc-His. The Xba I/Bam HIrestricted PCR fragment containing a 3′-truncated 76 kDa protein genewas ligated into the Xba I and Bam HI restricted plasmid pCA/Myc-His toproduce plasmid pCAD76kDa (FIG. 6).

[0189] The resulting plasmid, pCAD76kDa, was transferred byelectroporation into E. coli XL-1 blue (Stratagene) which was grown inLB broth containing 50 μg/ml of carbenicillin. The plasmid was isolatedby Endo Free Plasmid Giga Kit™ (Qiagen) large scale DNA purificationsystem. DNA concentration was determined by absorbance at 260 nm and theplasmid was verified after gel electrophoresis and Ethidium bromidestaining and comparison to molecular weight standards. The 5′ and 3′ends of the gene were verified by sequencing using a LiCor model 4000 LDNA sequencer and IRD-800 labelled primers.

Example 9

[0190] This example illustrates the immunization of mice to achieveprotection against an intranasal challenge of C. pneumoniae. Theprocedures are as described in Example 3 above, except that the DNAplasmid used for immunization contains the coding sequence of C.pneumoniae 3′-truncated 76 kDa protein, as described in Examples 7 and8.

[0191]FIG. 9 and Table 3 show that mice immunized i.n. and i.m. withpCAD76kDa had Chlamydial lung titers less than 2400 in 5 of 5 caseswhereas the range of values for control mice were 1800-23100 IFU/lung(mean 11811) and 16600-26100 IFU/lung (mean 22100) for sham immunizedwith saline or immunized with the unmodified vector respectively (Table2). The lack of protection with the unmodified vector confirms that DNAper se was not responsible for the observed protective effect. This isfurther supported by the results obtained for one additional plasmid DNAconstruct, pdagA, that failed to protect, and for which the mean lungtiters were similar to those obtained for saline-immunized control mice.The construct pdagA is identical to pCAD76kDa except that the nucleotidesequence encoding the 3′-truncated 76 kDa protein is replaced with aC.pneumoniae nucleotide sequence encoding the protein dagA. TABLE 3BACTERIAL LOAD (INCLUSION FORMING UNITS PER LUNG) IN THE LUNGS OF BALB/CMICE IMMUNIZED WITH VARIOUS DNA IMMUNIZATION CONSTRUCTS IMMUNIZINGCONSTRUCT MOUSE Saline Vector pdagA pCAD76kDa 1 17700 19900 16000 1700 23900 16600 500 2000 3 1800 24300 18500 2300 4 16400 26100 12800 2100 511700 23600 6400 600 6 23100 7 12000 8 5300 9 14400 10 18700 11 7300 128400 MEAN 11725 22100 10840 1740 SD 6567.71 3813.79 7344.59 673.05

[0192]

1 14 1 2156 DNA Chlamydia pneumoniae CDS (101)..(2053) 1 ataaaatctttaaaaacagg ctcgcattaa ttattagtga gagctttttt tttatttttt 60 ataataaaactaaaagattt ttattatttt ttgagttttt atg gtt aat cct att 115 Met Val Asn ProIle 1 5 ggt cca ggt cct ata gac gaa aca gaa cgc aca cct ccc gca gat ctt163 Gly Pro Gly Pro Ile Asp Glu Thr Glu Arg Thr Pro Pro Ala Asp Leu 1015 20 tct gct caa gga ttg gag gcg agt gca gca aat aag agt gcg gaa gct211 Ser Ala Gln Gly Leu Glu Ala Ser Ala Ala Asn Lys Ser Ala Glu Ala 2530 35 caa aga ata gca ggt gcg gaa gct aag cct aaa gaa tct aag acc gat259 Gln Arg Ile Ala Gly Ala Glu Ala Lys Pro Lys Glu Ser Lys Thr Asp 4045 50 tct gta gag cga tgg agc atc ttg cgt tct gca gtg aat gct ctc atg307 Ser Val Glu Arg Trp Ser Ile Leu Arg Ser Ala Val Asn Ala Leu Met 5560 65 agt ctg gca gat aag ctg ggt att gct tct agt aac agc tcg tct tct355 Ser Leu Ala Asp Lys Leu Gly Ile Ala Ser Ser Asn Ser Ser Ser Ser 7075 80 85 act agc aga tct gca gac gtg gac tca acg aca gcg acc gca cct acg403 Thr Ser Arg Ser Ala Asp Val Asp Ser Thr Thr Ala Thr Ala Pro Thr 9095 100 cct cct cca ccc acg ttt gat gat tat aag act caa gcg caa aca gct451 Pro Pro Pro Pro Thr Phe Asp Asp Tyr Lys Thr Gln Ala Gln Thr Ala 105110 115 tac gat act atc ttt acc tca aca tca cta gct gac ata cag gct gct499 Tyr Asp Thr Ile Phe Thr Ser Thr Ser Leu Ala Asp Ile Gln Ala Ala 120125 130 ttg gtg agc ctc cag gat gct gtc act aat ata aag gat aca gcg gct547 Leu Val Ser Leu Gln Asp Ala Val Thr Asn Ile Lys Asp Thr Ala Ala 135140 145 act gat gag gaa acc gca atc gct gcg gag tgg gaa act aag aat gcc595 Thr Asp Glu Glu Thr Ala Ile Ala Ala Glu Trp Glu Thr Lys Asn Ala 150155 160 165 gat gca gtt aaa gtt ggc gcg caa att aca gaa tta gcg aaa tatgct 643 Asp Ala Val Lys Val Gly Ala Gln Ile Thr Glu Leu Ala Lys Tyr Ala170 175 180 tcg gat aac caa gcg att ctt gac tct tta ggt aaa ctg act tccttc 691 Ser Asp Asn Gln Ala Ile Leu Asp Ser Leu Gly Lys Leu Thr Ser Phe185 190 195 gac ctc tta cag gct gct ctt ctc caa tct gta gca aac aat aacaaa 739 Asp Leu Leu Gln Ala Ala Leu Leu Gln Ser Val Ala Asn Asn Asn Lys200 205 210 gca gct gag ctt ctt aaa gag atg caa gat aac cca gta gtc ccaggg 787 Ala Ala Glu Leu Leu Lys Glu Met Gln Asp Asn Pro Val Val Pro Gly215 220 225 aaa acg cct gca att gct caa tct tta gtt gat cag aca gat gctaca 835 Lys Thr Pro Ala Ile Ala Gln Ser Leu Val Asp Gln Thr Asp Ala Thr230 235 240 245 gcg aca cag ata gag aaa gat gga aat gcg att agg gat gcatat ttt 883 Ala Thr Gln Ile Glu Lys Asp Gly Asn Ala Ile Arg Asp Ala TyrPhe 250 255 260 gca gga cag aac gct agt gga gct gta gaa aat gct aaa tctaat aac 931 Ala Gly Gln Asn Ala Ser Gly Ala Val Glu Asn Ala Lys Ser AsnAsn 265 270 275 agt ata agc aac ata gat tca gct aaa gca gca atc gct actgct aag 979 Ser Ile Ser Asn Ile Asp Ser Ala Lys Ala Ala Ile Ala Thr AlaLys 280 285 290 aca caa ata gct gaa gct cag aaa aag ttc ccc gac tct ccaatt ctt 1027 Thr Gln Ile Ala Glu Ala Gln Lys Lys Phe Pro Asp Ser Pro IleLeu 295 300 305 caa gaa gcg gaa caa atg gta ata cag gct gag aaa gat cttaaa aat 1075 Gln Glu Ala Glu Gln Met Val Ile Gln Ala Glu Lys Asp Leu LysAsn 310 315 320 325 atc aaa cct gca gat ggt tct gat gtt cca aat cca ggaact aca gtt 1123 Ile Lys Pro Ala Asp Gly Ser Asp Val Pro Asn Pro Gly ThrThr Val 330 335 340 gga ggc tcc aag caa caa gga agt agt att ggt agt attcgt gtt tcc 1171 Gly Gly Ser Lys Gln Gln Gly Ser Ser Ile Gly Ser Ile ArgVal Ser 345 350 355 atg ctg tta gat gat gct gaa aat gag acc gct tcc attttg atg tct 1219 Met Leu Leu Asp Asp Ala Glu Asn Glu Thr Ala Ser Ile LeuMet Ser 360 365 370 ggg ttt cgt cag atg att cac atg ttc aat acg gaa aatcct gat tct 1267 Gly Phe Arg Gln Met Ile His Met Phe Asn Thr Glu Asn ProAsp Ser 375 380 385 caa gct gcc caa cag gag ctc gca gca caa gct aga gcagcg aaa gcc 1315 Gln Ala Ala Gln Gln Glu Leu Ala Ala Gln Ala Arg Ala AlaLys Ala 390 395 400 405 gct gga gat gac agt gct gct gca gcg ctg gca gatgct cag aaa gct 1363 Ala Gly Asp Asp Ser Ala Ala Ala Ala Leu Ala Asp AlaGln Lys Ala 410 415 420 tta gaa gcg gct cta ggt aaa gct ggg caa caa cagggc ata ctc aat 1411 Leu Glu Ala Ala Leu Gly Lys Ala Gly Gln Gln Gln GlyIle Leu Asn 425 430 435 gct tta gga cag atc gct tct gct gct gtt gtg agcgca gga gtt cct 1459 Ala Leu Gly Gln Ile Ala Ser Ala Ala Val Val Ser AlaGly Val Pro 440 445 450 ccc gct gca gca agt tct ata ggg tca tct gta aaacag ctt tac aag 1507 Pro Ala Ala Ala Ser Ser Ile Gly Ser Ser Val Lys GlnLeu Tyr Lys 455 460 465 acc tca aaa tct aca ggt tct gat tat aaa aca cagata tca gca ggt 1555 Thr Ser Lys Ser Thr Gly Ser Asp Tyr Lys Thr Gln IleSer Ala Gly 470 475 480 485 tat gat gct tac aaa tcc atc aat gat gcc tatggt agg gca cga aat 1603 Tyr Asp Ala Tyr Lys Ser Ile Asn Asp Ala Tyr GlyArg Ala Arg Asn 490 495 500 gat gcg act cgt gat gtg ata aac aat gta agtacc ccc gct ctc aca 1651 Asp Ala Thr Arg Asp Val Ile Asn Asn Val Ser ThrPro Ala Leu Thr 505 510 515 cga tcc gtt cct aga gca cga aca gaa gct cgagga cca gaa aaa aca 1699 Arg Ser Val Pro Arg Ala Arg Thr Glu Ala Arg GlyPro Glu Lys Thr 520 525 530 gat caa gcc ctc gct agg gtg att tct ggc aatagc aga act ctt gga 1747 Asp Gln Ala Leu Ala Arg Val Ile Ser Gly Asn SerArg Thr Leu Gly 535 540 545 gat gtc tat agt caa gtt tcg gca cta caa tctgta atg cag atc atc 1795 Asp Val Tyr Ser Gln Val Ser Ala Leu Gln Ser ValMet Gln Ile Ile 550 555 560 565 cag tcg aat cct caa gcg aat aat gag gagatc aga caa aag ctt aca 1843 Gln Ser Asn Pro Gln Ala Asn Asn Glu Glu IleArg Gln Lys Leu Thr 570 575 580 tcg gca gtg aca aag cct cca cag ttt ggctat cct tat gtg caa ctt 1891 Ser Ala Val Thr Lys Pro Pro Gln Phe Gly TyrPro Tyr Val Gln Leu 585 590 595 tct aat gac tct aca cag aag ttc ata gctaaa tta gaa agt ttg ttt 1939 Ser Asn Asp Ser Thr Gln Lys Phe Ile Ala LysLeu Glu Ser Leu Phe 600 605 610 gct gaa gga tct agg aca gca gct gaa ataaaa gca ctt tcc ttt gaa 1987 Ala Glu Gly Ser Arg Thr Ala Ala Glu Ile LysAla Leu Ser Phe Glu 615 620 625 acg aac tcc ttg ttt att cag cag gtg ctggtc aat atc ggc tct cta 2035 Thr Asn Ser Leu Phe Ile Gln Gln Val Leu ValAsn Ile Gly Ser Leu 630 635 640 645 tat tct ggt tat ctc caa taacaacacctaagtgttcg tttggagaga 2083 Tyr Ser Gly Tyr Leu Gln 650 ttattatgtgctttggtaag gcctttgttg aggccttacc aacacactag aacgatcttc 2143 aataaataaaaga 2156 2 651 PRT Chlamydia pneumoniae 2 Met Val Asn Pro Ile Gly ProGly Pro Ile Asp Glu Thr Glu Arg Thr 1 5 10 15 Pro Pro Ala Asp Leu SerAla Gln Gly Leu Glu Ala Ser Ala Ala Asn 20 25 30 Lys Ser Ala Glu Ala GlnArg Ile Ala Gly Ala Glu Ala Lys Pro Lys 35 40 45 Glu Ser Lys Thr Asp SerVal Glu Arg Trp Ser Ile Leu Arg Ser Ala 50 55 60 Val Asn Ala Leu Met SerLeu Ala Asp Lys Leu Gly Ile Ala Ser Ser 65 70 75 80 Asn Ser Ser Ser SerThr Ser Arg Ser Ala Asp Val Asp Ser Thr Thr 85 90 95 Ala Thr Ala Pro ThrPro Pro Pro Pro Thr Phe Asp Asp Tyr Lys Thr 100 105 110 Gln Ala Gln ThrAla Tyr Asp Thr Ile Phe Thr Ser Thr Ser Leu Ala 115 120 125 Asp Ile GlnAla Ala Leu Val Ser Leu Gln Asp Ala Val Thr Asn Ile 130 135 140 Lys AspThr Ala Ala Thr Asp Glu Glu Thr Ala Ile Ala Ala Glu Trp 145 150 155 160Glu Thr Lys Asn Ala Asp Ala Val Lys Val Gly Ala Gln Ile Thr Glu 165 170175 Leu Ala Lys Tyr Ala Ser Asp Asn Gln Ala Ile Leu Asp Ser Leu Gly 180185 190 Lys Leu Thr Ser Phe Asp Leu Leu Gln Ala Ala Leu Leu Gln Ser Val195 200 205 Ala Asn Asn Asn Lys Ala Ala Glu Leu Leu Lys Glu Met Gln AspAsn 210 215 220 Pro Val Val Pro Gly Lys Thr Pro Ala Ile Ala Gln Ser LeuVal Asp 225 230 235 240 Gln Thr Asp Ala Thr Ala Thr Gln Ile Glu Lys AspGly Asn Ala Ile 245 250 255 Arg Asp Ala Tyr Phe Ala Gly Gln Asn Ala SerGly Ala Val Glu Asn 260 265 270 Ala Lys Ser Asn Asn Ser Ile Ser Asn IleAsp Ser Ala Lys Ala Ala 275 280 285 Ile Ala Thr Ala Lys Thr Gln Ile AlaGlu Ala Gln Lys Lys Phe Pro 290 295 300 Asp Ser Pro Ile Leu Gln Glu AlaGlu Gln Met Val Ile Gln Ala Glu 305 310 315 320 Lys Asp Leu Lys Asn IleLys Pro Ala Asp Gly Ser Asp Val Pro Asn 325 330 335 Pro Gly Thr Thr ValGly Gly Ser Lys Gln Gln Gly Ser Ser Ile Gly 340 345 350 Ser Ile Arg ValSer Met Leu Leu Asp Asp Ala Glu Asn Glu Thr Ala 355 360 365 Ser Ile LeuMet Ser Gly Phe Arg Gln Met Ile His Met Phe Asn Thr 370 375 380 Glu AsnPro Asp Ser Gln Ala Ala Gln Gln Glu Leu Ala Ala Gln Ala 385 390 395 400Arg Ala Ala Lys Ala Ala Gly Asp Asp Ser Ala Ala Ala Ala Leu Ala 405 410415 Asp Ala Gln Lys Ala Leu Glu Ala Ala Leu Gly Lys Ala Gly Gln Gln 420425 430 Gln Gly Ile Leu Asn Ala Leu Gly Gln Ile Ala Ser Ala Ala Val Val435 440 445 Ser Ala Gly Val Pro Pro Ala Ala Ala Ser Ser Ile Gly Ser SerVal 450 455 460 Lys Gln Leu Tyr Lys Thr Ser Lys Ser Thr Gly Ser Asp TyrLys Thr 465 470 475 480 Gln Ile Ser Ala Gly Tyr Asp Ala Tyr Lys Ser IleAsn Asp Ala Tyr 485 490 495 Gly Arg Ala Arg Asn Asp Ala Thr Arg Asp ValIle Asn Asn Val Ser 500 505 510 Thr Pro Ala Leu Thr Arg Ser Val Pro ArgAla Arg Thr Glu Ala Arg 515 520 525 Gly Pro Glu Lys Thr Asp Gln Ala LeuAla Arg Val Ile Ser Gly Asn 530 535 540 Ser Arg Thr Leu Gly Asp Val TyrSer Gln Val Ser Ala Leu Gln Ser 545 550 555 560 Val Met Gln Ile Ile GlnSer Asn Pro Gln Ala Asn Asn Glu Glu Ile 565 570 575 Arg Gln Lys Leu ThrSer Ala Val Thr Lys Pro Pro Gln Phe Gly Tyr 580 585 590 Pro Tyr Val GlnLeu Ser Asn Asp Ser Thr Gln Lys Phe Ile Ala Lys 595 600 605 Leu Glu SerLeu Phe Ala Glu Gly Ser Arg Thr Ala Ala Glu Ile Lys 610 615 620 Ala LeuSer Phe Glu Thr Asn Ser Leu Phe Ile Gln Gln Val Leu Val 625 630 635 640Asn Ile Gly Ser Leu Tyr Ser Gly Tyr Leu Gln 645 650 3 1852 DNA Chlamydiapneumoniae CDS (1)..(1749) 3 atg agt ctg gca gat aag ctg ggt att gct tctagt aac agc tcg tct 48 Met Ser Leu Ala Asp Lys Leu Gly Ile Ala Ser SerAsn Ser Ser Ser 1 5 10 15 tct act agc aga tct gca gac gtg gac tca acgaca gcg acc gca cct 96 Ser Thr Ser Arg Ser Ala Asp Val Asp Ser Thr ThrAla Thr Ala Pro 20 25 30 acg cct cct cca ccc acg ttt gat gat tat aag actcaa gcg caa aca 144 Thr Pro Pro Pro Pro Thr Phe Asp Asp Tyr Lys Thr GlnAla Gln Thr 35 40 45 gct tac gat act atc ttt acc tca aca tca cta gct gacata cag gct 192 Ala Tyr Asp Thr Ile Phe Thr Ser Thr Ser Leu Ala Asp IleGln Ala 50 55 60 gct ttg gtg agc ctc cag gat gct gtc act aat ata aag gataca gcg 240 Ala Leu Val Ser Leu Gln Asp Ala Val Thr Asn Ile Lys Asp ThrAla 65 70 75 80 gct act gat gag gaa acc gca atc gct gcg gag tgg gaa actaag aat 288 Ala Thr Asp Glu Glu Thr Ala Ile Ala Ala Glu Trp Glu Thr LysAsn 85 90 95 gcc gat gca gtt aaa gtt ggc gcg caa att aca gaa tta gcg aaatat 336 Ala Asp Ala Val Lys Val Gly Ala Gln Ile Thr Glu Leu Ala Lys Tyr100 105 110 gct tcg gat aac caa gcg att ctt gac tct tta ggt aaa ctg acttcc 384 Ala Ser Asp Asn Gln Ala Ile Leu Asp Ser Leu Gly Lys Leu Thr Ser115 120 125 ttc gac ctc tta cag gct gct ctt ctc caa tct gta gca aac aataac 432 Phe Asp Leu Leu Gln Ala Ala Leu Leu Gln Ser Val Ala Asn Asn Asn130 135 140 aaa gca gct gag ctt ctt aaa gag atg caa gat aac cca gta gtccca 480 Lys Ala Ala Glu Leu Leu Lys Glu Met Gln Asp Asn Pro Val Val Pro145 150 155 160 ggg aaa acg cct gca att gct caa tct tta gtt gat cag acagat gct 528 Gly Lys Thr Pro Ala Ile Ala Gln Ser Leu Val Asp Gln Thr AspAla 165 170 175 aca gcg aca cag ata gag aaa gat gga aat gcg att agg gatgca tat 576 Thr Ala Thr Gln Ile Glu Lys Asp Gly Asn Ala Ile Arg Asp AlaTyr 180 185 190 ttt gca gga cag aac gct agt gga gct gta gaa aat gct aaatct aat 624 Phe Ala Gly Gln Asn Ala Ser Gly Ala Val Glu Asn Ala Lys SerAsn 195 200 205 aac agt ata agc aac ata gat tca gct aaa gca gca atc gctact gct 672 Asn Ser Ile Ser Asn Ile Asp Ser Ala Lys Ala Ala Ile Ala ThrAla 210 215 220 aag aca caa ata gct gaa gct cag aaa aag ttc ccc gac tctcca att 720 Lys Thr Gln Ile Ala Glu Ala Gln Lys Lys Phe Pro Asp Ser ProIle 225 230 235 240 ctt caa gaa gcg gaa caa atg gta ata cag gct gag aaagat ctt aaa 768 Leu Gln Glu Ala Glu Gln Met Val Ile Gln Ala Glu Lys AspLeu Lys 245 250 255 aat atc aaa cct gca gat ggt tct gat gtt cca aat ccagga act aca 816 Asn Ile Lys Pro Ala Asp Gly Ser Asp Val Pro Asn Pro GlyThr Thr 260 265 270 gtt gga ggc tcc aag caa caa gga agt agt att ggt agtatt cgt gtt 864 Val Gly Gly Ser Lys Gln Gln Gly Ser Ser Ile Gly Ser IleArg Val 275 280 285 tcc atg ctg tta gat gat gct gaa aat gag acc gct tccatt ttg atg 912 Ser Met Leu Leu Asp Asp Ala Glu Asn Glu Thr Ala Ser IleLeu Met 290 295 300 tct ggg ttt cgt cag atg att cac atg ttc aat acg gaaaat cct gat 960 Ser Gly Phe Arg Gln Met Ile His Met Phe Asn Thr Glu AsnPro Asp 305 310 315 320 tct caa gct gcc caa cag gag ctc gca gca caa gctaga gca gcg aaa 1008 Ser Gln Ala Ala Gln Gln Glu Leu Ala Ala Gln Ala ArgAla Ala Lys 325 330 335 gcc gct gga gat gac agt gct gct gca gcg ctg gcagat gct cag aaa 1056 Ala Ala Gly Asp Asp Ser Ala Ala Ala Ala Leu Ala AspAla Gln Lys 340 345 350 gct tta gaa gcg gct cta ggt aaa gct ggg caa caacag ggc ata ctc 1104 Ala Leu Glu Ala Ala Leu Gly Lys Ala Gly Gln Gln GlnGly Ile Leu 355 360 365 aat gct tta gga cag atc gct tct gct gct gtt gtgagc gca gga gtt 1152 Asn Ala Leu Gly Gln Ile Ala Ser Ala Ala Val Val SerAla Gly Val 370 375 380 cct ccc gct gca gca agt tct ata ggg tca tct gtaaaa cag ctt tac 1200 Pro Pro Ala Ala Ala Ser Ser Ile Gly Ser Ser Val LysGln Leu Tyr 385 390 395 400 aag acc tca aaa tct aca ggt tct gat tat aaaaca cag ata tca gca 1248 Lys Thr Ser Lys Ser Thr Gly Ser Asp Tyr Lys ThrGln Ile Ser Ala 405 410 415 ggt tat gat gct tac aaa tcc atc aat gat gcctat ggt agg gca cga 1296 Gly Tyr Asp Ala Tyr Lys Ser Ile Asn Asp Ala TyrGly Arg Ala Arg 420 425 430 aat gat gcg act cgt gat gtg ata aac aat gtaagt acc ccc gct ctc 1344 Asn Asp Ala Thr Arg Asp Val Ile Asn Asn Val SerThr Pro Ala Leu 435 440 445 aca cga tcc gtt cct aga gca cga aca gaa gctcga gga cca gaa aaa 1392 Thr Arg Ser Val Pro Arg Ala Arg Thr Glu Ala ArgGly Pro Glu Lys 450 455 460 aca gat caa gcc ctc gct agg gtg att tct ggcaat agc aga act ctt 1440 Thr Asp Gln Ala Leu Ala Arg Val Ile Ser Gly AsnSer Arg Thr Leu 465 470 475 480 gga gat gtc tat agt caa gtt tcg gca ctacaa tct gta atg cag atc 1488 Gly Asp Val Tyr Ser Gln Val Ser Ala Leu GlnSer Val Met Gln Ile 485 490 495 act cag tcg aat cct caa gcg aat aat gaggag atc aga caa aag ctt 1536 Thr Gln Ser Asn Pro Gln Ala Asn Asn Glu GluIle Arg Gln Lys Leu 500 505 510 aca tcg gca gtg aca aag cct cca cag tttggc tat cct tat gtg caa 1584 Thr Ser Ala Val Thr Lys Pro Pro Gln Phe GlyTyr Pro Tyr Val Gln 515 520 525 ctt tct aat gac tct aca cag aag ttc atagct aaa tta gaa agt ttg 1632 Leu Ser Asn Asp Ser Thr Gln Lys Phe Ile AlaLys Leu Glu Ser Leu 530 535 540 ttt gct gaa gga tct agg aca gca gct gaaata aaa gca ctt tcc ttt 1680 Phe Ala Glu Gly Ser Arg Thr Ala Ala Glu IleLys Ala Leu Ser Phe 545 550 555 560 gaa acg aac tcc ttg ttt att cag caggtg ctg gtc aat atc ggc tct 1728 Glu Thr Asn Ser Leu Phe Ile Gln Gln ValLeu Val Asn Ile Gly Ser 565 570 575 cta tat tct ggt tat ctc caataacaacacc taagtgttcg tttggagaga 1779 Leu Tyr Ser Gly Tyr Leu Gln 580ttattatgtg ctttggtaag gcctttgttg aggccttacc aacacactag aacgatcttc 1839aataaataaa aga 1852 4 583 PRT Chlamydia pneumoniae 4 Met Ser Leu Ala AspLys Leu Gly Ile Ala Ser Ser Asn Ser Ser Ser 1 5 10 15 Ser Thr Ser ArgSer Ala Asp Val Asp Ser Thr Thr Ala Thr Ala Pro 20 25 30 Thr Pro Pro ProPro Thr Phe Asp Asp Tyr Lys Thr Gln Ala Gln Thr 35 40 45 Ala Tyr Asp ThrIle Phe Thr Ser Thr Ser Leu Ala Asp Ile Gln Ala 50 55 60 Ala Leu Val SerLeu Gln Asp Ala Val Thr Asn Ile Lys Asp Thr Ala 65 70 75 80 Ala Thr AspGlu Glu Thr Ala Ile Ala Ala Glu Trp Glu Thr Lys Asn 85 90 95 Ala Asp AlaVal Lys Val Gly Ala Gln Ile Thr Glu Leu Ala Lys Tyr 100 105 110 Ala SerAsp Asn Gln Ala Ile Leu Asp Ser Leu Gly Lys Leu Thr Ser 115 120 125 PheAsp Leu Leu Gln Ala Ala Leu Leu Gln Ser Val Ala Asn Asn Asn 130 135 140Lys Ala Ala Glu Leu Leu Lys Glu Met Gln Asp Asn Pro Val Val Pro 145 150155 160 Gly Lys Thr Pro Ala Ile Ala Gln Ser Leu Val Asp Gln Thr Asp Ala165 170 175 Thr Ala Thr Gln Ile Glu Lys Asp Gly Asn Ala Ile Arg Asp AlaTyr 180 185 190 Phe Ala Gly Gln Asn Ala Ser Gly Ala Val Glu Asn Ala LysSer Asn 195 200 205 Asn Ser Ile Ser Asn Ile Asp Ser Ala Lys Ala Ala IleAla Thr Ala 210 215 220 Lys Thr Gln Ile Ala Glu Ala Gln Lys Lys Phe ProAsp Ser Pro Ile 225 230 235 240 Leu Gln Glu Ala Glu Gln Met Val Ile GlnAla Glu Lys Asp Leu Lys 245 250 255 Asn Ile Lys Pro Ala Asp Gly Ser AspVal Pro Asn Pro Gly Thr Thr 260 265 270 Val Gly Gly Ser Lys Gln Gln GlySer Ser Ile Gly Ser Ile Arg Val 275 280 285 Ser Met Leu Leu Asp Asp AlaGlu Asn Glu Thr Ala Ser Ile Leu Met 290 295 300 Ser Gly Phe Arg Gln MetIle His Met Phe Asn Thr Glu Asn Pro Asp 305 310 315 320 Ser Gln Ala AlaGln Gln Glu Leu Ala Ala Gln Ala Arg Ala Ala Lys 325 330 335 Ala Ala GlyAsp Asp Ser Ala Ala Ala Ala Leu Ala Asp Ala Gln Lys 340 345 350 Ala LeuGlu Ala Ala Leu Gly Lys Ala Gly Gln Gln Gln Gly Ile Leu 355 360 365 AsnAla Leu Gly Gln Ile Ala Ser Ala Ala Val Val Ser Ala Gly Val 370 375 380Pro Pro Ala Ala Ala Ser Ser Ile Gly Ser Ser Val Lys Gln Leu Tyr 385 390395 400 Lys Thr Ser Lys Ser Thr Gly Ser Asp Tyr Lys Thr Gln Ile Ser Ala405 410 415 Gly Tyr Asp Ala Tyr Lys Ser Ile Asn Asp Ala Tyr Gly Arg AlaArg 420 425 430 Asn Asp Ala Thr Arg Asp Val Ile Asn Asn Val Ser Thr ProAla Leu 435 440 445 Thr Arg Ser Val Pro Arg Ala Arg Thr Glu Ala Arg GlyPro Glu Lys 450 455 460 Thr Asp Gln Ala Leu Ala Arg Val Ile Ser Gly AsnSer Arg Thr Leu 465 470 475 480 Gly Asp Val Tyr Ser Gln Val Ser Ala LeuGln Ser Val Met Gln Ile 485 490 495 Thr Gln Ser Asn Pro Gln Ala Asn AsnGlu Glu Ile Arg Gln Lys Leu 500 505 510 Thr Ser Ala Val Thr Lys Pro ProGln Phe Gly Tyr Pro Tyr Val Gln 515 520 525 Leu Ser Asn Asp Ser Thr GlnLys Phe Ile Ala Lys Leu Glu Ser Leu 530 535 540 Phe Ala Glu Gly Ser ArgThr Ala Ala Glu Ile Lys Ala Leu Ser Phe 545 550 555 560 Glu Thr Asn SerLeu Phe Ile Gln Gln Val Leu Val Asn Ile Gly Ser 565 570 575 Leu Tyr SerGly Tyr Leu Gln 580 5 1456 DNA Chlamydia pneumoniae CDS (101)..(1456) 5ataaaatctt taaaaacagg ctcgcattaa ttattagtga gagctttttt tttatttttt 60ataataaaac taaaagattt ttattatttt ttgagttttt atg gtt aat cct att 115 MetVal Asn Pro Ile 1 5 ggt cca ggt cct ata gac gaa aca gaa cgc aca cct cccgca gat ctt 163 Gly Pro Gly Pro Ile Asp Glu Thr Glu Arg Thr Pro Pro AlaAsp Leu 10 15 20 tct gct caa gga ttg gag gcg agt gca gca aat aag agt gcggaa gct 211 Ser Ala Gln Gly Leu Glu Ala Ser Ala Ala Asn Lys Ser Ala GluAla 25 30 35 caa aga ata gca ggt gcg gaa gct aag cct aaa gaa tct aag accgat 259 Gln Arg Ile Ala Gly Ala Glu Ala Lys Pro Lys Glu Ser Lys Thr Asp40 45 50 tct gta gag cga tgg agc atc ttg cgt tct gca gtg aat gct ctc atg307 Ser Val Glu Arg Trp Ser Ile Leu Arg Ser Ala Val Asn Ala Leu Met 5560 65 agt ctg gca gat aag ctg ggt att gct tct agt aac agc tcg tct tct355 Ser Leu Ala Asp Lys Leu Gly Ile Ala Ser Ser Asn Ser Ser Ser Ser 7075 80 85 act agc aga tct gca gac gtg gac tca acg aca gcg acc gca cct acg403 Thr Ser Arg Ser Ala Asp Val Asp Ser Thr Thr Ala Thr Ala Pro Thr 9095 100 cct cct cca ccc acg ttt gat gat tat aag act caa gcg caa aca gct451 Pro Pro Pro Pro Thr Phe Asp Asp Tyr Lys Thr Gln Ala Gln Thr Ala 105110 115 tac gat act atc ttt acc tca aca tca cta gct gac ata cag gct gct499 Tyr Asp Thr Ile Phe Thr Ser Thr Ser Leu Ala Asp Ile Gln Ala Ala 120125 130 ttg gtg agc ctc cag gat gct gtc act aat ata aag gat aca gcg gct547 Leu Val Ser Leu Gln Asp Ala Val Thr Asn Ile Lys Asp Thr Ala Ala 135140 145 act gat gag gaa acc gca atc gct gcg gag tgg gaa act aag aat gcc595 Thr Asp Glu Glu Thr Ala Ile Ala Ala Glu Trp Glu Thr Lys Asn Ala 150155 160 165 gat gca gtt aaa gtt ggc gcg caa att aca gaa tta gcg aaa tatgct 643 Asp Ala Val Lys Val Gly Ala Gln Ile Thr Glu Leu Ala Lys Tyr Ala170 175 180 tcg gat aac caa gcg att ctt gac tct tta ggt aaa ctg act tccttc 691 Ser Asp Asn Gln Ala Ile Leu Asp Ser Leu Gly Lys Leu Thr Ser Phe185 190 195 gac ctc tta cag gct gct ctt ctc caa tct gta gca aac aat aacaaa 739 Asp Leu Leu Gln Ala Ala Leu Leu Gln Ser Val Ala Asn Asn Asn Lys200 205 210 gca gct gag ctt ctt aaa gag atg caa gat aac cca gta gtc ccaggg 787 Ala Ala Glu Leu Leu Lys Glu Met Gln Asp Asn Pro Val Val Pro Gly215 220 225 aaa acg cct gca att gct caa tct tta gtt gat cag aca gat gctaca 835 Lys Thr Pro Ala Ile Ala Gln Ser Leu Val Asp Gln Thr Asp Ala Thr230 235 240 245 gcg aca cag ata gag aaa gat gga aat gcg att agg gat gcatat ttt 883 Ala Thr Gln Ile Glu Lys Asp Gly Asn Ala Ile Arg Asp Ala TyrPhe 250 255 260 gca gga cag aac gct agt gga gct gta gaa aat gct aaa tctaat aac 931 Ala Gly Gln Asn Ala Ser Gly Ala Val Glu Asn Ala Lys Ser AsnAsn 265 270 275 agt ata agc aac ata gat tca gct aaa gca gca atc gct actgct aag 979 Ser Ile Ser Asn Ile Asp Ser Ala Lys Ala Ala Ile Ala Thr AlaLys 280 285 290 aca caa ata gct gaa gct cag aaa aag ttc ccc gac tct ccaatt ctt 1027 Thr Gln Ile Ala Glu Ala Gln Lys Lys Phe Pro Asp Ser Pro IleLeu 295 300 305 caa gaa gcg gaa caa atg gta ata cag gct gag aaa gat cttaaa aat 1075 Gln Glu Ala Glu Gln Met Val Ile Gln Ala Glu Lys Asp Leu LysAsn 310 315 320 325 atc aaa cct gca gat ggt tct gat gtt cca aat cca ggaact aca gtt 1123 Ile Lys Pro Ala Asp Gly Ser Asp Val Pro Asn Pro Gly ThrThr Val 330 335 340 gga ggc tcc aag caa caa gga agt agt att ggt agt attcgt gtt tcc 1171 Gly Gly Ser Lys Gln Gln Gly Ser Ser Ile Gly Ser Ile ArgVal Ser 345 350 355 atg ctg tta gat gat gct gaa aat gag acc gct tcc attttg atg tct 1219 Met Leu Leu Asp Asp Ala Glu Asn Glu Thr Ala Ser Ile LeuMet Ser 360 365 370 ggg ttt cgt cag atg att cac atg ttc aat acg gaa aatcct gat tct 1267 Gly Phe Arg Gln Met Ile His Met Phe Asn Thr Glu Asn ProAsp Ser 375 380 385 caa gct gcc caa cag gag ctc gca gca caa gct aga gcagcg aaa gcc 1315 Gln Ala Ala Gln Gln Glu Leu Ala Ala Gln Ala Arg Ala AlaLys Ala 390 395 400 405 gct gga gat gac agt gct gct gca gcg ctg gca gatgct cag aaa gct 1363 Ala Gly Asp Asp Ser Ala Ala Ala Ala Leu Ala Asp AlaGln Lys Ala 410 415 420 tta gaa gcg gct cta ggt aaa gct ggg caa caa cagggc ata ctc aat 1411 Leu Glu Ala Ala Leu Gly Lys Ala Gly Gln Gln Gln GlyIle Leu Asn 425 430 435 gct tta gga cag atc gct tct gct gct gtt gtg agcgca gga gta 1456 Ala Leu Gly Gln Ile Ala Ser Ala Ala Val Val Ser Ala GlyVal 440 445 450 6 452 PRT Chlamydia pneumoniae 6 Met Val Asn Pro Ile GlyPro Gly Pro Ile Asp Glu Thr Glu Arg Thr 1 5 10 15 Pro Pro Ala Asp LeuSer Ala Gln Gly Leu Glu Ala Ser Ala Ala Asn 20 25 30 Lys Ser Ala Glu AlaGln Arg Ile Ala Gly Ala Glu Ala Lys Pro Lys 35 40 45 Glu Ser Lys Thr AspSer Val Glu Arg Trp Ser Ile Leu Arg Ser Ala 50 55 60 Val Asn Ala Leu MetSer Leu Ala Asp Lys Leu Gly Ile Ala Ser Ser 65 70 75 80 Asn Ser Ser SerSer Thr Ser Arg Ser Ala Asp Val Asp Ser Thr Thr 85 90 95 Ala Thr Ala ProThr Pro Pro Pro Pro Thr Phe Asp Asp Tyr Lys Thr 100 105 110 Gln Ala GlnThr Ala Tyr Asp Thr Ile Phe Thr Ser Thr Ser Leu Ala 115 120 125 Asp IleGln Ala Ala Leu Val Ser Leu Gln Asp Ala Val Thr Asn Ile 130 135 140 LysAsp Thr Ala Ala Thr Asp Glu Glu Thr Ala Ile Ala Ala Glu Trp 145 150 155160 Glu Thr Lys Asn Ala Asp Ala Val Lys Val Gly Ala Gln Ile Thr Glu 165170 175 Leu Ala Lys Tyr Ala Ser Asp Asn Gln Ala Ile Leu Asp Ser Leu Gly180 185 190 Lys Leu Thr Ser Phe Asp Leu Leu Gln Ala Ala Leu Leu Gln SerVal 195 200 205 Ala Asn Asn Asn Lys Ala Ala Glu Leu Leu Lys Glu Met GlnAsp Asn 210 215 220 Pro Val Val Pro Gly Lys Thr Pro Ala Ile Ala Gln SerLeu Val Asp 225 230 235 240 Gln Thr Asp Ala Thr Ala Thr Gln Ile Glu LysAsp Gly Asn Ala Ile 245 250 255 Arg Asp Ala Tyr Phe Ala Gly Gln Asn AlaSer Gly Ala Val Glu Asn 260 265 270 Ala Lys Ser Asn Asn Ser Ile Ser AsnIle Asp Ser Ala Lys Ala Ala 275 280 285 Ile Ala Thr Ala Lys Thr Gln IleAla Glu Ala Gln Lys Lys Phe Pro 290 295 300 Asp Ser Pro Ile Leu Gln GluAla Glu Gln Met Val Ile Gln Ala Glu 305 310 315 320 Lys Asp Leu Lys AsnIle Lys Pro Ala Asp Gly Ser Asp Val Pro Asn 325 330 335 Pro Gly Thr ThrVal Gly Gly Ser Lys Gln Gln Gly Ser Ser Ile Gly 340 345 350 Ser Ile ArgVal Ser Met Leu Leu Asp Asp Ala Glu Asn Glu Thr Ala 355 360 365 Ser IleLeu Met Ser Gly Phe Arg Gln Met Ile His Met Phe Asn Thr 370 375 380 GluAsn Pro Asp Ser Gln Ala Ala Gln Gln Glu Leu Ala Ala Gln Ala 385 390 395400 Arg Ala Ala Lys Ala Ala Gly Asp Asp Ser Ala Ala Ala Ala Leu Ala 405410 415 Asp Ala Gln Lys Ala Leu Glu Ala Ala Leu Gly Lys Ala Gly Gln Gln420 425 430 Gln Gly Ile Leu Asn Ala Leu Gly Gln Ile Ala Ser Ala Ala ValVal 435 440 445 Ser Ala Gly Val 450 7 2238 DNA Chlamydia pneumoniae CDS(766)..(2235) 7 atgacaaaaa aacattatgc ttgggttgta gaagggattc tcaatcgtttgcctaaacag 60 ttttttgtga aatgtagtgt tgtcgactgg aacacattcg ttccttcagaaacctccact 120 acagaaaaag ctgctacaaa cgctatgaaa tacaaatact gtgtttggcagtggctcgtc 180 ggaaagcata gtcaggttcc ttggatcaat ggacagaaaa agcctctatatctttatgga 240 gctttcttaa tgaacccttt agcaaaggct acgaagacta cgttaaatggaaaagaaaac 300 ctagcttggt ttattggagg aactttaggg ggactcagaa aagctggagactggtctgcc 360 acagtacgtt atgagtatgt cgaagccttg tcggttccag aaatagatgtttcagggatt 420 ggccgtggta atttattaaa gttttggttc gcccaagcaa ttgctgctaactatgatcct 480 aaagaggcta atggttttac aaattataaa ggattttccg ctctatatatgtatggcatc 540 acagattctc tatcattcag agcttatggg gcttactcca aaccagcaaacgataaactc 600 ggcagtgatt ttactttccg aaagtttgat ctaggtataa tttcagcgttttaagtcaaa 660 ttttaataaa atctttaaaa acaggctcgc attaattatt agtgagagctttttttttat 720 tttttataat aaaactaaaa gatttttatt attttttgag ttttt atg gttaat cct 777 Met Val Asn Pro 1 att ggt cca ggt cct ata gac gaa aca gaacgc aca cct ccc gca gat 825 Ile Gly Pro Gly Pro Ile Asp Glu Thr Glu ArgThr Pro Pro Ala Asp 5 10 15 20 ctt tct gct caa gga ttg gag gcg agt gcagca aat aag agt gcg gaa 873 Leu Ser Ala Gln Gly Leu Glu Ala Ser Ala AlaAsn Lys Ser Ala Glu 25 30 35 gct caa aga ata gca ggt gcg gaa gct aag cctaaa gaa tct aag acc 921 Ala Gln Arg Ile Ala Gly Ala Glu Ala Lys Pro LysGlu Ser Lys Thr 40 45 50 gat tct gta gag cga tgg agc atc ttg cgt tct gcagtg aat gct ctc 969 Asp Ser Val Glu Arg Trp Ser Ile Leu Arg Ser Ala ValAsn Ala Leu 55 60 65 atg agt ctg gca gat aag ctg ggt att gct tct agt aacagc tcg tct 1017 Met Ser Leu Ala Asp Lys Leu Gly Ile Ala Ser Ser Asn SerSer Ser 70 75 80 tct act agc aga tct gca gac gtg gac tca acg aca gcg accgca cct 1065 Ser Thr Ser Arg Ser Ala Asp Val Asp Ser Thr Thr Ala Thr AlaPro 85 90 95 100 acg cct cct cca ccc acg ttt gat gat tat aag act caa gcgcaa aca 1113 Thr Pro Pro Pro Pro Thr Phe Asp Asp Tyr Lys Thr Gln Ala GlnThr 105 110 115 gct tac gat act atc ttt acc tca aca tca cta gct gac atacag gct 1161 Ala Tyr Asp Thr Ile Phe Thr Ser Thr Ser Leu Ala Asp Ile GlnAla 120 125 130 gct ttg gtg agc ctc cag gat gct gtc act aat ata aag gataca gcg 1209 Ala Leu Val Ser Leu Gln Asp Ala Val Thr Asn Ile Lys Asp ThrAla 135 140 145 gct act gat gag gaa acc gca atc gct gcg gag tgg gaa actaag aat 1257 Ala Thr Asp Glu Glu Thr Ala Ile Ala Ala Glu Trp Glu Thr LysAsn 150 155 160 gcc gat gca gtt aaa gtt ggc gcg caa att aca gaa tta gcgaaa tat 1305 Ala Asp Ala Val Lys Val Gly Ala Gln Ile Thr Glu Leu Ala LysTyr 165 170 175 180 gct tcg gat aac caa gcg att ctt gac tct tta ggt aaactg act tcc 1353 Ala Ser Asp Asn Gln Ala Ile Leu Asp Ser Leu Gly Lys LeuThr Ser 185 190 195 ttc gac ctc tta cag gct gct ctt ctc caa tct gta gcaaac aat aac 1401 Phe Asp Leu Leu Gln Ala Ala Leu Leu Gln Ser Val Ala AsnAsn Asn 200 205 210 aaa gca gct gag ctt ctt aaa gag atg caa gat aac ccagta gtc cca 1449 Lys Ala Ala Glu Leu Leu Lys Glu Met Gln Asp Asn Pro ValVal Pro 215 220 225 ggg aaa acg cct gca att gct caa tct tta gtt gat cagaca gat gct 1497 Gly Lys Thr Pro Ala Ile Ala Gln Ser Leu Val Asp Gln ThrAsp Ala 230 235 240 aca gcg aca cag ata gag aaa gat gga aat gcg att agggat gca tat 1545 Thr Ala Thr Gln Ile Glu Lys Asp Gly Asn Ala Ile Arg AspAla Tyr 245 250 255 260 ttt gca gga cag aac gct agt gga gct gta gaa aatgct aaa tct aat 1593 Phe Ala Gly Gln Asn Ala Ser Gly Ala Val Glu Asn AlaLys Ser Asn 265 270 275 aac agt ata agc aac ata gat tca gct aaa gca gcaatc gct act gct 1641 Asn Ser Ile Ser Asn Ile Asp Ser Ala Lys Ala Ala IleAla Thr Ala 280 285 290 aag aca caa ata gct gaa gct cag aaa aag ttc cccgac tct cca att 1689 Lys Thr Gln Ile Ala Glu Ala Gln Lys Lys Phe Pro AspSer Pro Ile 295 300 305 ctt caa gaa gcg gaa caa atg gta ata cag gct gagaaa gat ctt aaa 1737 Leu Gln Glu Ala Glu Gln Met Val Ile Gln Ala Glu LysAsp Leu Lys 310 315 320 aat atc aaa cct gca gat ggt tct gat gtt cca aatcca gga act aca 1785 Asn Ile Lys Pro Ala Asp Gly Ser Asp Val Pro Asn ProGly Thr Thr 325 330 335 340 gtt gga ggc tcc aag caa caa gga agt agt attggt agt att cgt gtt 1833 Val Gly Gly Ser Lys Gln Gln Gly Ser Ser Ile GlySer Ile Arg Val 345 350 355 tcc atg ctg tta gat gat gct gaa aat gag accgct tcc att ttg atg 1881 Ser Met Leu Leu Asp Asp Ala Glu Asn Glu Thr AlaSer Ile Leu Met 360 365 370 tct ggg ttt cgt cag atg att cac atg ttc aatacg gaa aat cct gat 1929 Ser Gly Phe Arg Gln Met Ile His Met Phe Asn ThrGlu Asn Pro Asp 375 380 385 tct caa gct gcc caa cag gag ctc gca gca caagct aga gca gcg aaa 1977 Ser Gln Ala Ala Gln Gln Glu Leu Ala Ala Gln AlaArg Ala Ala Lys 390 395 400 gcc gct gga gat gac agt gct gct gca gcg ctggca gat gct cag aaa 2025 Ala Ala Gly Asp Asp Ser Ala Ala Ala Ala Leu AlaAsp Ala Gln Lys 405 410 415 420 gct tta gaa gcg gct cta ggt aaa gct gggcaa caa cag ggc ata ctc 2073 Ala Leu Glu Ala Ala Leu Gly Lys Ala Gly GlnGln Gln Gly Ile Leu 425 430 435 aat gct tta gga cag atc gct tct gct gctgtt gtg agc gca gga gta 2121 Asn Ala Leu Gly Gln Ile Ala Ser Ala Ala ValVal Ser Ala Gly Val 440 445 450 ctc ccg ctg cag caa gtt cta tgg atc cgagct cgg tac caa gct tac 2169 Leu Pro Leu Gln Gln Val Leu Trp Ile Arg AlaArg Tyr Gln Ala Tyr 455 460 465 gta gaa caa aaa ctc atc tca gaa gag gatctg aat agc gcc gtc gac 2217 Val Glu Gln Lys Leu Ile Ser Glu Glu Asp LeuAsn Ser Ala Val Asp 470 475 480 cat cat cat cat cat cat tga 2238 His HisHis His His His 485 490 8 490 PRT Chlamydia pneumoniae 8 Met Val Asn ProIle Gly Pro Gly Pro Ile Asp Glu Thr Glu Arg Thr 1 5 10 15 Pro Pro AlaAsp Leu Ser Ala Gln Gly Leu Glu Ala Ser Ala Ala Asn 20 25 30 Lys Ser AlaGlu Ala Gln Arg Ile Ala Gly Ala Glu Ala Lys Pro Lys 35 40 45 Glu Ser LysThr Asp Ser Val Glu Arg Trp Ser Ile Leu Arg Ser Ala 50 55 60 Val Asn AlaLeu Met Ser Leu Ala Asp Lys Leu Gly Ile Ala Ser Ser 65 70 75 80 Asn SerSer Ser Ser Thr Ser Arg Ser Ala Asp Val Asp Ser Thr Thr 85 90 95 Ala ThrAla Pro Thr Pro Pro Pro Pro Thr Phe Asp Asp Tyr Lys Thr 100 105 110 GlnAla Gln Thr Ala Tyr Asp Thr Ile Phe Thr Ser Thr Ser Leu Ala 115 120 125Asp Ile Gln Ala Ala Leu Val Ser Leu Gln Asp Ala Val Thr Asn Ile 130 135140 Lys Asp Thr Ala Ala Thr Asp Glu Glu Thr Ala Ile Ala Ala Glu Trp 145150 155 160 Glu Thr Lys Asn Ala Asp Ala Val Lys Val Gly Ala Gln Ile ThrGlu 165 170 175 Leu Ala Lys Tyr Ala Ser Asp Asn Gln Ala Ile Leu Asp SerLeu Gly 180 185 190 Lys Leu Thr Ser Phe Asp Leu Leu Gln Ala Ala Leu LeuGln Ser Val 195 200 205 Ala Asn Asn Asn Lys Ala Ala Glu Leu Leu Lys GluMet Gln Asp Asn 210 215 220 Pro Val Val Pro Gly Lys Thr Pro Ala Ile AlaGln Ser Leu Val Asp 225 230 235 240 Gln Thr Asp Ala Thr Ala Thr Gln IleGlu Lys Asp Gly Asn Ala Ile 245 250 255 Arg Asp Ala Tyr Phe Ala Gly GlnAsn Ala Ser Gly Ala Val Glu Asn 260 265 270 Ala Lys Ser Asn Asn Ser IleSer Asn Ile Asp Ser Ala Lys Ala Ala 275 280 285 Ile Ala Thr Ala Lys ThrGln Ile Ala Glu Ala Gln Lys Lys Phe Pro 290 295 300 Asp Ser Pro Ile LeuGln Glu Ala Glu Gln Met Val Ile Gln Ala Glu 305 310 315 320 Lys Asp LeuLys Asn Ile Lys Pro Ala Asp Gly Ser Asp Val Pro Asn 325 330 335 Pro GlyThr Thr Val Gly Gly Ser Lys Gln Gln Gly Ser Ser Ile Gly 340 345 350 SerIle Arg Val Ser Met Leu Leu Asp Asp Ala Glu Asn Glu Thr Ala 355 360 365Ser Ile Leu Met Ser Gly Phe Arg Gln Met Ile His Met Phe Asn Thr 370 375380 Glu Asn Pro Asp Ser Gln Ala Ala Gln Gln Glu Leu Ala Ala Gln Ala 385390 395 400 Arg Ala Ala Lys Ala Ala Gly Asp Asp Ser Ala Ala Ala Ala LeuAla 405 410 415 Asp Ala Gln Lys Ala Leu Glu Ala Ala Leu Gly Lys Ala GlyGln Gln 420 425 430 Gln Gly Ile Leu Asn Ala Leu Gly Gln Ile Ala Ser AlaAla Val Val 435 440 445 Ser Ala Gly Val Leu Pro Leu Gln Gln Val Leu TrpIle Arg Ala Arg 450 455 460 Tyr Gln Ala Tyr Val Glu Gln Lys Leu Ile SerGlu Glu Asp Leu Asn 465 470 475 480 Ser Ala Val Asp His His His His HisHis 485 490 9 43 DNA Artificial Sequence Description of ArtificialSequence Primer 9 ataagaatgc ggccgccacc atggttaatc ctattggtcc agg 43 1035 DNA Artificial Sequence Description of Artificial Sequence Primer 10gcgccggatc ccttggagat aaccagaata tagag 35 11 43 DNA Artificial SequenceDescription of Artificial Sequence Primer 11 ataagaatgc ggccgccaccatgagtctgg cagataagct ggg 43 12 32 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 12 gcgccggatc ccttggagat aaccagaata ta 3213 38 DNA Artificial Sequence Description of Artificial Sequence Primer13 gctctagacc gccatgacaa aaaaacatta tgcttggg 38 14 28 DNA ArtificialSequence Description of Artificial Sequence Primer 14 cgggatccatagaacttgct gcagcggg 28

1. A nucleic acid molecule comprising a nucleic acid sequence whichencodes a polypeptide selected from any of: (a) SEQ ID No: 2; (b) SEQ IDNo. 4; (c) SEQ ID No. 6; (d) an immunogenic fragment comprising at least12 consecutive amino acids from a polypeptide of (a); and (e) apolypeptide of any one of (a) to (d) which has been modified to improveits immunogenicity, wherein said modified polypeptide is at least 75%identical in amino acid sequence to the corresponding polypeptide of anyone of (a) to (d).
 2. A nucleic acid molecule comprising a nucleic acidsequence selected from any of: (a) SEQ ID No: 1; (b) SEQ ID No: 3; (c)SEQ ID No: 5; (d) a sequence which encodes a polypeptide encoded by anyone of SEQ ID Nos: 1, 3 and 5; (e) a sequence comprising at least 38consecutive nucleotides from any one of the nucleic acid sequences of(a) to (d); and (f) a sequence which encodes a polypeptide which is atleast 75% identical in amino acid sequence to the polypeptides encodedby any one of SEQ ID Nos: 1, 3 and
 5. 3. A nucleic acid moleculecomprising a nucleic acid sequence which is anti-sense to the nucleicacid molecule of claim
 1. 4. A nucleic acid molecule comprising anucleic acid sequence which encodes a fusion protein, said fusionprotein comprising a polypeptide encoded by a nucleic acid moleculeaccording to claim 1 and an additional polypeptide.
 5. The nucleic acidmolecule of claim 4 wherein the additional polypeptide is a heterologoussignal peptide.
 6. The nucleic acid molecule of claim 4 wherein theadditional polypeptide has adjuvant activity.
 7. The nucleic acidmolecule according to claim 1, operatively linked to one or moreexpression control sequences.
 8. A vaccine comprising at least one firstnucleic acid according to claim 1, and a vaccine vector wherein eachfirst nucleic acid is expressed as a polypeptide, the vaccine optionallycomprising a second nucleic acid encoding an additional polypeptidewhich enhances the immune response to the polypeptide expressed by saidfirst nucleic acid.
 9. The vaccine of claim 8 wherein the second nucleicacid encodes an additional Chlamydia polypeptide.
 10. A pharmaceuticalcomposition comprising a nucleic acid according to claim 1 and apharmaceutically acceptable carrier.
 11. A pharmaceutical compositioncomprising a vaccine according to claim 8 and a pharmaceuticallyacceptable carrier.
 12. A unicellular host transformed with the nucleicacid molecule of claim
 7. 13. A nucleic acid probe of 5 to 100nucleotides which hybridizes under stringent conditions to the nucleicacid molecule of SEQ ID No: 1, or to a homolog or complementary oranti-sense sequence of said nucleic acid molecule.
 14. A primer of 10 to40 nucleotides which hybridizes under stringent conditions to thenucleic acid molecules of SEQ ID No: 1, or to a homolog or complementaryor anti-sense sequence of said nucleic acid molecule.
 15. A polypeptidecomprising an amino acid sequence selected from any of: (a) SEQ ID No:2; (b) SEQ ID No: 4; (c) SEQ ID No: 6; (d) an immunogenic fragmentcomprising at least 12 consecutive amino acids from a polypeptide of(a); and (e) a polypeptide of any one of (a) to (d) which has beenmodified to improve its immunogenicity, wherein said modifiedpolypeptide is at least 75% identical in amino acid sequence to thecorresponding polypeptide of any one of (a) to (d).
 16. A fusionpolypeptide comprising the polypeptide of claim 15 and an additionalpolypeptide.
 17. The fusion polypeptide of claim 16 wherein theadditional polypeptide is a heterologous signal peptide.
 18. The fusionprotein of claim 16 wherein the additional polypeptide has adjuvantactivity.
 19. A method for producing a polypeptide of claim 15,comprising the step of culturing a unicellular host according to claim12.
 20. An antibody against the polypeptide of claim
 15. 21. A vaccinecomprising at least one first polypeptide according to claim 15 and apharmaceutically acceptable carrier, optionally comprising a secondpolypeptide which enhances the immune response to the first polypeptide.22. The vaccine of claim 21 wherein the second polypeptide comprises anadditional Chlamydia polypeptide.
 23. A pharmaceutical compositioncomprising a polypeptide according to claim 15 and a pharmaceuticallyacceptable carrier.
 24. A pharmaceutical composition comprising avaccine according to claim 21 and a pharmaceutically acceptable carrier.25. A pharmaceutical composition comprising an antibody according toclaim 20 and a pharmaceutically acceptable carrier.
 26. A method forpreventing or treating Chlamydia infection using the nucleic acid ofclaim
 1. 27. A method for preventing or treating Chlamydia infectionusing the vaccine of claim
 8. 28. A method for preventing or treatingChlamydia infection using the pharmaceutical composition of claim 10.29. A method for preventing or treating Chlamydia infection using thepolypeptide of claim
 15. 30. A method for preventing or treatingChlamydia infection using the antibody of claim
 20. 31. A method ofdetecting Chlamydia infection comprising the step of assaying a bodyfluid of a mammal to be tested with the nucleic acid of claim
 1. 32. Amethod of detecting Chlamydia infection comprising the step of assayinga body fluid of a mammal to be tested with the polypeptide of claim 15.33. A method of detecting Chlamydia infection comprising the step ofassaying a body fluid of a mammal to be tested with the antibody ofclaim
 20. 34. A method for identifying the polypeptide of claim 15 whichinduces an immune response effective to prevent or lessen the severityof Chlamydia infection in a mammal previously immunized withpolypeptide, comprising the steps of: (a) immunizing a mouse with thepolypeptide; and (b) inoculating the immunized mouse with Chlamydia;wherein the polypeptide which prevents or lessens the severity ofChlamydia infection in the immunized mouse compared to a non-immunizedcontrol mouse is identified.
 35. An expression plasmid selected from thegroup consisting of pCACPNM555a, pCAI555 and pCAD76kDa.
 36. A nucleicacid molecule selected from the group consisting of SEQ ID Nos: 1, 3, 5and
 7. 37. A polypeptide selected from the group consisting of SEQ IDNos: 2, 4, 6 and
 8. 38. An isolated 76 kDa protein from Chlamydia.